CN118460688A - Establishment of a method for simultaneous detection of single-cell transcriptome and multiple targeted proteins in paraformaldehyde-fixed cells - Google Patents

Abstract

The invention discloses establishment of a single-cell transcriptome of paraformaldehyde fixed cells and a simultaneous detection method of multiple target proteins. The invention provides a single-cell multi-group chemical detection method of paraformaldehyde fixed cells, which comprises the following steps: A. fixing and membrane-penetrating treatment is carried out on single cells by using 4% of PFA as a fixing agent and 0.1% of Triton as a membrane-penetrating agent; B. lysing the cells after the fixation and membrane permeation treatment in a single-cell operation system, establishing a library, sequencing, and realizing single-cell multi-group chemical detection of the paraformaldehyde fixed cells; the lysis is performed in a high throughput single cell microplate system of the single cell handling system. The invention determines an antibody binding scheme capable of simultaneously binding cell membrane surface proteins, cytoplasmic phosphorylated proteins and nuclear transcription factors, and can better keep RNA intact; the method of cell de-crosslinking after paraformaldehyde treatment was determined and compatible with single cell operating systems.

Description

Establishment of a method for simultaneous detection of single-cell transcriptome and multiple targeted proteins in paraformaldehyde-fixed cells

Technical Field

The present invention belongs to high-throughput single-cell sequencing technology, and relates to the establishment of a method for simultaneously detecting single-cell transcriptome and multiple targeted proteins of paraformaldehyde-fixed cells.

Background Art

Studies have linked single-cell transcriptomics data to targeted protein detection using DNA barcode antibodies. Stoeckius et al. published CITE-seq technology in Nature Methods in 2017. Based on the idea of DNA barcodes, the method simultaneously measured membrane surface proteins and transcriptomes. The method reads out the information of the antibody sequence through sequencing to trace the expression of the protein, thereby exploring the relationship between RNA transcripts and proteins. At the same time, Peterson et al. published REAP-seq (RNA expression and protein sequencing assay). The basic principles of the two are the same. Chung et al. published inCITE-seq (intranuclear cellular indexing of transcriptomes and epitopes) technology in Nature Methods in 2021. Based on CITE-seq technology, single cell nuclei were extracted, low-concentration formaldehyde and NP-40 were used to fix the cell nuclei and permeabilize the membrane, and antibodies coupled to oligonucleotides were used to bind to transcription factors, so as to read the mRNA and transcription factor expression of the cell nucleus through sequencing. Mimitou et al. used the bridging method to recombinant antibody tags and developed ASAP-seq (ATAC with select antigen profiling by sequencing), which can simultaneously analyze the chromatin accessible regions and protein levels of single cells. This method also uses low-concentration formaldehyde and NP-40 to fix the membrane, and then marks the chromatin open regions with Tn5 transposase with sequencing adapters. Subsequently, combined with 10x Genomics' Multiome product, it can perform multimodal analysis of chromatin accessibility, gene expression, and cell membrane surface proteins from the same cell, named DOGMA-seq (a variant of CITE-seq, allowing co-measurement of chromatin accessibility, gene expression and protein from the same cells). This work was published in Nature Biotechnology in 2021.

In summary, the above technologies suggest the scalability based on the idea of DNA barcoding. The inCITE-seq technology directly extracts single cell nuclei based on 1% FA and 0.2% NP-40, so the information of mature cytoplasmic mRNA, membrane surface proteins and cytoplasmic proteins will be lost. Although ASAP-seq can measure the information of chromatin accessible regions and proteins in a single cell, it loses the mRNA information. DOGMA-seq loses the information of cytoplasmic and nuclear localized proteins.

Therefore, existing methods are limited to only being able to analyze membrane surface proteins or only being able to analyze some proteins within the cell, and cannot achieve comprehensive coverage of membrane surface proteins, cytoplasmic phosphorylated proteins, and nuclear localized proteins. In order to determine the expression of each localized protein within the cell, the cells need to be fixed and permeabilized. Current cell fixation and permeabilization methods hinder the efficiency of mRNA detection and limit the ability to simultaneously measure intracellular protein expression and transcriptional profiles in single cells.

Summary of the invention

The purpose of the present invention is to establish a method for simultaneously detecting single cell transcriptome and multiple targeted proteins of paraformaldehyde-fixed cells.

In a first aspect, the present invention provides a single-cell multi-omics detection method for paraformaldehyde-fixed cells, comprising the following steps:

A. Use 4% PFA as fixative and 0.1% Triton as permeabilization agent to fix and permeabilize single cells.

B. Lysing the fixed and permeabilized cells in a single-cell operating system, followed by library construction and sequencing to achieve single-cell multi-omics detection of paraformaldehyde-fixed cells;

The lysis is performed in a high-throughput single-cell microplate system of the single-cell operating system.

In the above method, if the single-cell multi-omics detection is mRNA whole transcriptomics detection, the method includes step A and step B;

Further, in an embodiment of the present invention, if the single-cell multi-omics detection is mRNA whole transcriptomics detection, the method comprises the following steps:

1) Obtain single cell suspension;

2) using 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent to fix and permeabilize the single cells to obtain permeabilized cells (corresponding to step A of the first aspect);

3) performing high-throughput single-cell detection on the permeabilized cells in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the steps of adding magnetic beads for capturing nucleic acids, cell lysis, and enriching magnetic beads to obtain single-cell mRNA, and then using the single-cell mRNA to construct an mRNA full transcriptome library (corresponding to step B of the first aspect);

4) Sequencing the mRNA whole transcriptome library to obtain mRNA sequences, and realizing single-cell multi-omics detection (mRNA whole transcriptome detection) (corresponding to step B of the first aspect).

If the single-cell multi-omics detection is a membrane surface protein detection, then before the step A, the following step A-1 is also included: labeling the single cell with one or more antibody-oligos that bind to the membrane surface protein to be detected to obtain a membrane surface protein labeled cell;

Further, in an embodiment of the present invention, if the single-cell multi-omics detection is membrane surface protein detection, the method comprises the following steps:

1) Obtain a single cell, one or more antibodies-oligos that bind to the target protein to be detected;

The target protein includes a single cell membrane surface protein;

2) labeling the single cell with one or more antibody-oligos that bind to the membrane surface protein to be detected to obtain a membrane surface protein labeled cell (corresponding to step A-1);

3) using 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent to fix and permeabilize the cells labeled with membrane surface proteins to obtain permeabilized cells (corresponding to step A of the first aspect);

4) performing high-throughput single-cell detection on the permeabilized cells in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the steps of adding magnetic beads for capturing nucleic acids, cell lysis, and enriching magnetic beads to obtain all antibody-oligos connected to single-cell target proteins, and then using all antibody-oligos connected to single-cell target proteins to construct an antibody library (corresponding to step B of the first aspect);

6) Sequencing the antibody library to obtain the expression of the oligo sequence, thereby knowing the expression of the target protein bound by the antibody corresponding to the oligo sequence, and realizing single-cell multi-omics detection (membrane surface protein detection) (corresponding to step B of the first aspect).

If the single-cell multi-omics detection is a non-membrane surface protein detection, then between the steps A and B, the following step A-2 is also included: labeling the fixed permeabilized cells with one or more antibodies-oligos that bind to the non-membrane surface protein to be detected to obtain labeled cells;

The non-membrane surface protein is a cytoplasmic protein, a phosphorylated protein and/or a nuclear transcription factor;

Further, in an embodiment of the present invention, if the single cell multi-omics detection is non-membrane surface protein detection, the method comprises the following steps:

1) Obtain a single cell, one or more antibodies-oligos that bind to the target protein to be detected;

The target proteins include single cell cytoplasmic proteins, single cell phosphorylated proteins and/or single cell nuclear transcription factors;

2) using 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent to fix and permeabilize the single cell to obtain a permeabilized cell (corresponding to step A of the first aspect);

3) labeling the permeabilized cells with one or more single cell cytoplasmic protein antibody-oligo and/or one or more single cell intracellular protein antibody-oligo to obtain a labeled single cell suspension (corresponding to step A-2);

The intracellular proteins mentioned above include phosphorylated proteins or nuclear transcription factors;

4) subjecting the labeled single cell suspension to high-throughput single cell detection in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the steps of adding magnetic beads for capturing nucleic acids, cell lysis, and enriching magnetic beads to obtain all antibody-oligos connected to single cell target proteins, and then using all antibody-oligos connected to single cell target proteins to construct an antibody library (corresponding to step B of the first aspect);

5) Sequencing the antibody library to obtain the expression of the oligo sequence, thereby knowing the expression of the target protein bound by the antibody corresponding to the oligo sequence, and realizing single-cell multi-omics detection (non-membrane surface protein detection) (corresponding to step B of the first aspect).

The above-mentioned single-cell multi-omics detection can be a single detection or a combination of detections. The combination detection can be performed by merging the corresponding single detection schemes. The details are as follows:

If the single-cell multi-omics detection is mRNA full transcriptomics detection and membrane surface protein detection, the method includes A-1, A and B;

Further, in an embodiment of the present invention, if the single-cell multi-omics detection is mRNA whole transcriptomics detection and membrane surface protein detection, the method comprises the following steps:

1) Obtain a single cell, one or more antibodies-oligos that bind to the target protein to be detected;

The target protein includes a single cell membrane surface protein;

2) labeling the single cell with one or more antibody-oligos that bind to the membrane surface protein to be detected to obtain a membrane surface protein labeled cell (corresponding to step A-1);

3) using 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent to fix and permeabilize the cells labeled with membrane surface proteins to obtain permeabilized cells (corresponding to step A of the first aspect);

4) subjecting the permeabilized cells to high-throughput single-cell detection in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the steps of adding magnetic beads for capturing nucleic acids, cell lysis, and enriching magnetic beads to obtain single-cell mRNA and all antibody-oligos connected to single-cell target proteins, and then using the single-cell mRNA and all antibody-oligos connected to single-cell target proteins to construct an mRNA full transcriptome library and an antibody library, respectively (corresponding to step B of the first aspect);

5) Sequencing the mRNA full transcriptome library and/or antibody library to obtain the expression of the mRNA sequence and/or oligo sequence, and knowing the expression of the target protein bound by the antibody corresponding to the oligo sequence from the expression of the oligo sequence, so as to realize single-cell multi-omics detection (mRNA full transcriptome detection and membrane surface protein detection) (corresponding to step B of the first aspect).

If the single-cell multi-omics detection is mRNA whole transcriptomics detection and non-membrane surface protein detection, the method includes A, A-2 and B;

Furthermore, if the single-cell multi-omics detection is mRNA whole transcriptomics detection and non-membrane surface protein detection, the method comprises the following steps:

1) Obtain a single cell, one or more antibodies-oligos that bind to the target protein to be detected;

The target proteins include single cell cytoplasmic proteins, single cell phosphorylated proteins and/or single cell nuclear transcription factors;

2) using 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent to fix and permeabilize the single cell to obtain a permeabilized cell (corresponding to step A of the first aspect);

3) labeling the permeabilized cells with one or more single cell cytoplasmic protein antibody-oligo and/or one or more single cell intracellular protein antibody-oligo to obtain a labeled single cell suspension (corresponding to step A-2);

The intracellular proteins mentioned above include phosphorylated proteins or nuclear transcription factors;

4) subjecting the labeled single cell suspension to high-throughput single cell detection in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the steps of adding magnetic beads for capturing nucleic acids, cell lysis, and enriching magnetic beads to obtain single cell mRNA and all antibody-oligos connected to single cell target proteins, and then using the single cell mRNA and all antibody-oligos connected to single cell target proteins to construct an mRNA full transcriptome library and an antibody library, respectively (corresponding to step B of the first aspect);

5) Sequencing the mRNA whole transcriptome library and/or antibody library to obtain the expression of the mRNA sequence and/or oligo sequence, and knowing the expression of the target protein bound by the antibody corresponding to the oligo sequence from the expression of the oligo sequence, so as to realize single-cell multi-omics detection (mRNA whole transcriptome detection and non-membrane surface protein detection) (corresponding to step B of the first aspect).

If the single-cell multi-omics detection is mRNA whole transcriptomics detection, membrane surface protein detection and non-membrane surface protein detection, the method includes A-1, A, A-2 and B.

Further, in an embodiment of the present invention, if the single-cell multi-omics detection is mRNA whole transcriptomics detection, membrane surface protein detection and non-membrane surface protein detection, the method comprises the following steps:

1) Obtain a single cell, one or more antibodies-oligos that bind to the target protein to be detected;

The target proteins include single cell membrane surface proteins, single cell cytoplasmic proteins, single cell phosphorylated proteins and/or single cell nuclear transcription factors;

2) labeling the single cell with one or more antibody-oligos that bind to the membrane surface protein to be detected to obtain a membrane surface protein labeled cell (corresponding to step A-1);

3) using 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent to fix and permeabilize the cells labeled with membrane surface proteins to obtain permeabilized cells (corresponding to step A of the first aspect);

4) labeling the permeabilized cells with one or more single cell cytoplasmic protein antibody-oligo and/or one or more single cell intracellular protein antibody-oligo to obtain a labeled single cell suspension (corresponding to step A-2);

The intracellular proteins mentioned above include phosphorylated proteins or nuclear transcription factors;

5) subjecting the labeled single cell suspension to high-throughput single cell detection in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the steps of adding magnetic beads for capturing nucleic acids, cell lysis, and enriching magnetic beads to obtain single cell mRNA and all antibody-oligos connected to single cell target proteins, and then using the single cell mRNA and all antibody-oligos connected to single cell target proteins to construct an mRNA full transcriptome library and an antibody library, respectively (corresponding to step B of the first aspect);

6) Sequencing the mRNA full transcriptome library and/or antibody library to obtain the expression of the mRNA sequence and/or oligo sequence, and knowing the expression of the target protein bound by the antibody corresponding to the oligo sequence from the expression of the oligo sequence, so as to realize single-cell multi-omics detection (mRNA full transcriptome detection, membrane surface protein detection and non-membrane surface protein detection) (corresponding to step B of the first aspect).

In the above method, each antibody-oligo is a conjugate obtained by connecting the target protein antibody to be detected with the oligo sequence through a click chemistry reaction of trans-cyclooctene (TCO) as a dienophile and S-tetrazine, the number of oligos coupled to the antibody is 2-3, the connection efficiency reaches 100%, and there is no free oligo sequence;

In the above antibody-oligo preparation method, the amount of antibody and oligo added is 30 pmol of oligo to 1 μg of antibody.

The oligo sequence includes a poly A terminal structure that can be captured by magnetic beads carrying oligo-dT, a barcode sequence for distinguishing different antibodies, a molecular identifier UMI, and a specific sequence for PCR amplification.

The Triton mentioned above in the embodiment of the present invention is Triton X-100.

In the above method, the single-cell operating system is a BD Rhapsody single-cell operating system;

The high-throughput single-cell microplate system is the BD Rhapsody system.

In the above method, the step B comprises the following steps: performing high-throughput single-cell detection on the fixed permeabilized cells in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, the following steps are performed in sequence: adding magnetic beads for capturing nucleic acids, cell lysis and enrichment of magnetic beads to obtain single-cell mRNA/or all antibody-oligos connected to proteins, and then using the single-cell mRNA and/or all antibody-oligos connected to proteins to construct an mRNA full transcriptome library and/or an antibody library respectively; sequencing to achieve single-cell multi-omics detection of paraformaldehyde-fixed cells;

Alternatively, one single cell and one magnetic bead are added to each microwell of the BD Rhapsody system;

Alternatively, the nucleic acid is mRNA and all antibody-oligos from a single cell.

Furthermore, the above high-throughput single-cell detection specifically includes the following steps in sequence: activating the microplate, adding the labeled single-cell suspension, adding magnetic beads for capturing nucleic acids, cell lysis, enriching the magnetic beads, obtaining cell mRNA and antibody-oligo linked to the target protein, and then constructing an mRNA full transcriptome library and/or an antibody library with the cell mRNA and/or the antibody-oligo linked to the target protein, and then sequencing the constructed libraries respectively;

In the above method, the magnetic beads used to capture nucleic acid are magnetic beads with oligo-dT, and the oligo-dT in the magnetic beads captures the antibody of protein-polyA in the oligo;

Alternatively, the lysis solution used for cell lysis is a lysis buffer containing proteinase K.

In the above method, the concentration of proteinase K in the lysis buffer containing proteinase K added to each microwell is 75mAU/mL, and the added volume is 100ul;

Or, in the cell lysis, the step of adding mineral oil is further included after adding the lysis buffer containing proteinase K;

Alternatively, the enriched magnetic beads are recovered by anhydrous ethanol.

In the above method, the mineral oil is vapor lock of Qiagen;

The mineral oil is added 25 seconds after the lysis buffer containing proteinase K is added;

The lysis time is 5 minutes at room temperature.

In the above method, the antibody-oligo of each protein is an antibody-oligo of a protein that binds to SSB.

The method for preparing the antibody-oligo after binding to SSB is as follows:

E.coli single-stranded DNA binding protein (EcoSSB) was incubated with antibody-oligo at 37 degrees for 30 minutes.

Furthermore, the incubation system is as follows: 25ul system: 10X NEB 4buffer 2.5ul; antibody-oligo titration amount; EcoSSB#Promega 1ul; the rest is filled with _H2O .

In the above method, in step A-2), the labeling is performed in a buffer containing saturated dextran sulfate.

In the above, the formula of the buffer solution containing saturated dextran sulfate is as follows: 1% BSA, 0.05% dextran amine, 1 U/mL RNase, and the remainder is RNase-free PBS.

The single-cell multi-omics detection includes transcriptome level detection, membrane surface protein detection, cytoplasmic protein detection, phosphorylated protein detection and/or nuclear transcription factor detection.

In a second aspect, the present invention provides the use of 4% paraformaldehyde as a fixative and 0.1% Triton as a membrane permeabilizing agent in fixing single cells.

In a third aspect, the present invention provides an application of step B in the method described in the first aspect in detecting the multi-omics state of a single cell after fixation with 4% PFA;

In the above application, the single-cell multi-omics status includes transcriptome level, membrane surface proteins, cytoplasmic proteins, phosphorylated proteins and/or nuclear transcription factors.

In a fourth aspect, the present invention provides 4% paraformaldehyde as a single cell fixative and 0.1% Triton as a single cell permeabilizing agent, and application of step B in the method described in the first aspect in single cell multi-omics detection.

The present invention adopts the following scheme:

(1) A fixation and permeabilization protocol was determined based on 4% PFA as a fixative for 5 minutes and 0.1% Triton as a permeabilization agent for 10 minutes. The gold standard flow cytometry method verified that this method had a good antibody binding effect on membrane surface proteins, phosphorylated proteins, and transcription factors.

(2) We explored the method of de-crosslinking cells after paraformaldehyde treatment, selected a high-throughput single-cell microplate system, and solved how to be compatible with the single-cell operating system. Taking the BD Rhapsody single-cell microplate system as an example, the existing single-cell operation process does not support de-crosslinking during the cell lysis step. Therefore, based on the existing operation process, after the cells are loaded onto the chip, the lysis buffer (BD lysis buffer plus 40U/ml proteinase K, the volume ratio of the two is 7:1) is added, and after standing at room temperature for 25 seconds, a layer of mineral oil (Qiagen #981611) is then spread, and the operation is incubated at room temperature for 5 minutes, and the capture magnetic beads are recovered using anhydrous ethanol.

(3) Based on the click chemistry reaction of trans-cyclooctene and tetrazine, an antibody coupled with an oligonucleotide sequence tag (hereinafter referred to as antibody-oligo) was prepared. SDS-PAGE showed that the antibody-oligonucleotide tag connection efficiency met the standard. The excess unbound oligonucleotide was removed using a method based on ammonium sulfate protein precipitation. Vertical electrophoresis showed that the antibody purification efficiency of the coupled tag sequence met the standard;

(4) Determine the antibody-oligo incubation system to reduce the background non-specificity of single-cell protein detection. First, use E. coli single-stranded DNA binding protein (EcoSSB) to incubate with antibody-oligo at 37 degrees for 30 minutes (25ul system: 10X NEB 4buffer 2.5ul; antibody-oligo titration amount; EcoSSB#Promega 1ul; the rest is filled with H2O). Directly blocking the charge of the antibody oligonucleotide may significantly improve the background signal and reduce non-specific binding. Secondly, when the antibody is labeled with the target protein, a blocking solution (1% BSA, 0.05% dextran amine, 1U/mL RNaseinhibitor in RNase-free PBS) is used to further reduce the electrostatic interaction between the negatively charged DNA oligonucleotide and the positively charged molecules of the cell (such as histones), thereby minimizing the non-specific background of the antibody-oligo. The present invention is then used to evaluate the detection sensitivity and quantitative accuracy of membrane surface proteins, intracellular phosphorylated proteins, and nuclear localization proteins.

Therefore, the present invention has the following advantages:

(1) An antibody binding scheme was identified that can simultaneously bind to cell membrane surface proteins, cytoplasmic phosphorylated proteins, and nuclear transcription factors, and can better preserve RNA integrity; a method for de-crosslinking cells after paraformaldehyde treatment was determined, which is compatible with the single-cell operating system. At the cell line level, single-cell transcriptome evaluation of fixed and permeabilized cell lines retained 98% of gene expression levels compared to fresh samples. At the primary cell level, spleen cells that were fixed and permeabilized retained 88% of gene expression levels compared to fresh samples, which is better than existing data.

(2) The electrochemical reaction of trans-cyclooctene and S-tetrazine was used to successfully prepare and purify antibodies coupled with DNA tags. Based on this, the present invention can accurately quantify the expression of CD3 and CD4 proteins on the membrane surface, detect the response of intracellular phosphorylated proteins, and accurately quantify the information of cytoplasmic/nuclear localized GFP proteins with different expression levels, which is equivalent to the flow cytometry results.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the technical process of the present invention, in which (A) indicates that the design of DNA barcode (oligo) includes the following four aspects: poly A terminal structure that can be captured by magnetic beads with oligo-dT; barcode sequence that distinguishes antibodies; unique molecular identifier UMI (Unique Molecular Identifier) used to control amplification deviation; and sequence that can be used for PCR amplification. Antibody-oligo is incubated and combined with cell surface protein; (B) cells are fixed, cell antigenicity is maintained, membrane is perforated, and cytoplasm and nuclear protein antibody-oligo is labeled; (C) single cells are loaded into microporous chips, and sequences containing poly T and cellbarcode capture mRNA released by cell lysis and antibody-oligo targeting proteins.

FIG. 2 shows the labeling of cytoplasmic protein ZAP70 and nuclear transcription factor FoxP3 under the conditions of 5-minute treatment with different fixatives and 10-minute permeabilization with 0.1% Triton.

FIG. 3 shows the labeling of phosphorylated p38 protein by the determined fixed permeabilization method.

FIG. 4 is a diagram for verifying the antibody-oligo connection, purification efficiency, and specificity of antigen recognition.

FIG5 shows the RNA retention of fixed permeabilized cells by INT-seq.

FIG6 is the quantitative results of membrane surface proteins by INT-seq.

Figure 7 is the response of INT-seq to detect phosphorylated P38 in HEK-293T cells; (A) Flow cytometry histogram shows the expression level of phosphorylated P38 in the Anisomycin unstimulated group and the stimulated group; (B) The expression level of phosphorylated P38 in the Anisomycin unstimulated group and the stimulated group was determined by INT-seq.

Figure 8 shows the detection effect of INT-seq on the gradient expression of cytoplasmic/nuclear localized GFP; (A) HEK-293T transfected with cytoplasmic GFP fluorescent protein; (B) Flow cytometry sorting of three groups of cells with high, medium and low expression of GFP protein; (C) Single cell system INT-seq detection of cytoplasmic GFP expression in three groups of cells sorted; (D) HEK-293T transfected with nuclear localized GFP fluorescent protein; (E) Flow cytometry sorting of three groups of cells with high, medium and low expression of GFP protein; (F) Single cell system INT-seq detection of nuclear GFP expression in three groups of cells sorted.

DETAILED DESCRIPTION

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The method of the present invention (hereinafter referred to as INT-seq: an INTegrated technology to simultaneously profile single-cell multiplexed proteins and transcriptome by SEQuencing) has the following reaction principles:

Based on the detection method of single-cell transcriptome and the idea of DNA barcode, antibodies with special oligonucleotide tags (hereinafter referred to as antibody-oligo) are added to the existing single-cell transcriptome. Antibody-oligo binds to the target protein, and the antibody sequence is read out through sequencing to trace the protein. The design of oligo (DNA barcode) includes the following four aspects: the poly A terminal structure that can be captured by the magnetic beads with oligo-dT; the barcode sequence that distinguishes the antibody; the unique molecular identifier UMI (Unique Molecular Identifier) used to control amplification deviation; and the sequence that can be used for PCR amplification.

The flow chart of the method of the present invention is shown in Figure 1. The design of the DNA barcode includes the following four aspects: a poly A terminal structure that can be captured by a magnetic bead with oligo-dT; a barcode sequence that distinguishes antibodies; a unique molecular identifier UMI (Unique Molecular Identifier) used to control amplification deviation; and a sequence that can be used for PCR amplification; in the figure, (A) antibody-oligo is incubated and combined with cell surface protein; (B) cells are fixed, the antigenicity of the cells is maintained, the membrane is perforated, cytoplasm and nuclear protein antibody-oligo is labeled, and unbound antibody-oligo is washed away; (C) a single cell is loaded into a microporous chip, a cell lysis solution containing proteinase K is added, mineral oil is spread, and the lysis is carried out at room temperature for 5 minutes, and the antibody-oligo and the poly A at the end of the mRNA are captured by the poly T on the magnetic beads, and then downstream analysis is performed.

The specific process is as follows: After the cell surface protein antibody-oligo binds to the cell surface protein, the cell is fixed to maintain the antigenicity of the cell, the membrane is perforated to allow the cytoplasmic antibody-oligo and/or nuclear protein antibody-oligo to enter the cell for labeling, and the unbound antibody-oligo is washed away. Then, the single cell is loaded onto the machine, and tens of thousands of cells are loaded into 200,000 microwells to ensure that each microwell captures one cell. Cell lysis and cross-linking removal need to be completed in the microwell. The poly A in different protein antibody-oligo and the poly A at the end of mRNA are captured and reverse transcribed by the poly T on the magnetic beads. After cDNA amplification, antibody-derived oligonucleotide amplification sequences (amplified antibody-derived tags, ADTs) and transcriptome amplification sequences are obtained. The transcriptome and antibody-derived oligonucleotide amplification sequences (amplified antibody-derived tags, ADTs) can be used to construct independent sequencing libraries according to their lengths, and then downstream analysis is performed.

The PBS in the following examples is 1x, free of calcium and magnesium, free of RNase, and has a pH value of 7.4, purchased from Beijing Regen Biotechnology Co., Ltd., Cat# NH0012.

Some of the methods in the following embodiments are as follows:

1. Commercial cytoplasmic protein staining method (hereinafter referred to as commercial reagent 1)

Use eBioscience ^TM IC Fixation Buffer (eBioscience #00-8222-49). Buffer and Solution Preparation: Dilute 10x Permeabilization Buffer with double distilled water to prepare 1x Permeabilization Buffer Working Solution; IC Fixation Buffer is 2x, no dilution is required.

(1) Preparing single cell suspension;

(2) staining for cell surface markers;

(3) After the final wash, discard the supernatant and pulse vortex the sample to completely dissociate cell clumps, usually leaving about 100 μL residual volume;

(4) Add 100 μL IC fixative to fix the cells, then pulse vortex to mix;

(5) Incubate at room temperature for 20-60 minutes, protected from light;

(6) Add 2 mL of 1x permeabilization solution, centrifuge at 400-600 g for 5 minutes at room temperature, and discard the supernatant;

(7) Repeat step 6;

(8) Resuspend the cell pellet in 100 μL of 1x fresh permeabilization solution, add flow cytometry antibodies according to the reagent manufacturer's recommended amount to detect the expression of intracellular antigens, and incubate at room temperature or 4 degrees in the dark for 20-60 minutes;

(9) Add 2 mL of 1x permeabilization buffer, centrifuge at 400-600 x g for 5 minutes at room temperature, and discard the supernatant;

(10) Repeat step 10;

(11) Resuspend the stained cells in an appropriate volume of FACS buffer;

(12) Detect the fluorescence expression of the sample by flow cytometer.

2. Commercialized nuclear protein Foxp3 staining method (hereinafter referred to as commercial reagent 2)

Use eBioscience ^TM Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (eBioscience #00-5521-00). Buffer and Solution Preparation: Mix Fixation/Permeabilization Concentrate and Fixation/Permeabilization Diluent in a 1:3 ratio to prepare a fresh working solution. Dilute 10x Permeabilization Buffer with double distilled water to prepare 1x Permeabilization Buffer Working Solution.

(1) Prepare a single cell suspension as described above;

(2) labeling cell surface with flow cytometry antibodies, such as CD3 and CD4, incubating on ice for 30 min, and then washing;

(3) After the last wash, 100 μL of residual PBS needs to be retained;

(4) Add 1 mL of fresh fixation/permeabilization working solution to each centrifuge tube and resuspend;

(5) Incubate at 4°C for 30 min in the dark, then centrifuge;

(6) Add 1 mL of 1x fresh permeabilization buffer, centrifuge the sample at 500 g for 3-5 minutes at room temperature, and discard the supernatant;

(7) Resuspend the pellet in the remaining 100 μL of permeabilization buffer;

(8) Add 2 μL of 2% normal mouse/rat serum directly to the cells for blocking and incubate at room temperature for 15 min;

(9) Without washing, the recommended amount of flow cytometry antibody can be directly added to detect intracellular antigens, and incubated at room temperature in the dark for more than 30 minutes. Then, 2 mL of 1x permeabilization buffer was added to each tube to terminate the reaction. The sample was centrifuged at 500 g for 5 minutes at room temperature and the supernatant was removed.

(10) Repeat step 10;

(11) Resuspend the treated cells in an appropriate volume of FACS buffer;

(12) Detect the fluorescence expression of the sample by flow cytometer.

3. The single cell data processing process is as follows

The raw reads were processed by the BD Rhapsody whole transcriptome analysis pipeline. The processing included filtering by read quality, read annotation, molecular annotation, putative cells, and generation of single-cell expression matrices. Appropriate thresholds were designed based on the characteristics of the data to exclude the following types of low-quality cells: (1) cells with too low or too high gene counts. Cells with too low gene counts contain incomplete expression information, while cells with too high gene counts may be dimorphic cells; (2) cells with too high a proportion of mitochondrial genes, which are in poor condition or dead. Principal component analysis (PCA) was used for dimensionality reduction, combined with the nearest neighbor method or the Harmony method to batch correct for repetitions and abiotic confounding factors between different times. After the principal components were determined, cluster analysis was performed using the t-distributed stochastic neighbor embedding (t-SNE) or UMAP (Uniform Manifold Approximation and Projection for Dimension Reduction). Based on existing knowledge, cell subpopulations were identified and the types of each cell subpopulation were defined.

Example 1: Exploring the Optimal Single Cell Immobilization and Permeabilization Method

1. Effect of single cell fixation and permeabilization method for membrane surface protein detection

In order to explore a fixation permeabilization scheme that has a good antibody binding effect on membrane surface proteins, cytoplasmic phosphorylated proteins, and nuclear transcription factors, the corresponding commercial fixation permeabilization reagents were used as positive controls, and the eBioscience ^TM IC fixation permeabilization solution specifically for cytoplasmic protein detection (hereinafter referred to as commercial buffer 1) was used to label cytoplasmic proteins (the method is described above), and the eBioscience ^TM fixation/permeabilization concentrate specifically for nuclear localization protein detection (hereinafter referred to as commercial buffer 2) was used to label intracellular nuclear transcription factors (the method is described above), and the expression of specific proteins was detected by flow cytometry.

Spleen cells were selected as experimental samples. ZAP70 plays an important role in regulating adaptive immune responses and is usually expressed on the inner side of the membrane surface of T cells and natural killer cells as a cytoplasmic protein. Foxp3 was identified as a specific transcription factor that controls Treg cell differentiation as an intracellular nuclear transcription factor. Therefore, the membrane surface protein CD3, as well as ZAP70 protein and Foxp3 protein, which are proteins with clear intracellular expression and accurate localization, were selected to evaluate the effect of fixed permeabilization agent labeling target protein.

(1) Preparation of mouse spleen single cell suspension

The mice were killed by cervical dislocation, and the hair on the surface of the mice was sprayed with 75% alcohol, and the abdominal cavity was opened; spleen cells were obtained: the spleen was removed with forceps, and washed with PBS, and the spleen was squeezed with the flat end of a 1mL syringe plunger to release spleen cells. The obtained cell suspension (suspended in PBS) was filtered through a 70μm cell mesh into a 15mL centrifuge tube. Centrifuge at 500g for 5min, 4 degrees, discard the supernatant, and collect the precipitate; the precipitate was then subjected to red blood cell lysis: 1-2mL of red blood cell lysis solution (depending on the amount of cells) was used to lyse the red blood cells at room temperature for 2min, and filtered with a mesh, and 3-5 times the volume of PBS was used to terminate the red blood cell lysis, and then the precipitate was collected by centrifugation at 500g for 5min; the washing was repeated twice with PBS to obtain a single cell suspension of mouse spleen;

(2) Add 2 μL of membrane surface protein CD3 antibody (FITC anti-mouse CD3ε, Biolegend, Cat#100306) to 100 μL of spleen single cell suspension (2×10 ⁶ cells/ml) obtained in step (1) above, and react at 4°C for 30 min to label the membrane surface protein; centrifuge at 4°C, 500 g for 5 min, and collect the precipitate (cell pellet);

(3) The precipitate collected in step (2) was resuspended in 1 ml of 4% PFA (Biosharp Cat#BL539A) or 1.5% PFA solution (1.5% PFA is obtained by diluting 4% PFA with water), fixed at room temperature for 5 min, then washed twice, centrifuged at 4°C, 500 g for 5 min, and the precipitate was collected;

(4) The precipitate collected in step (3) was resuspended in 1 ml of 0.1% (volume percentage) TritonX-100 solution (solvent: PBS) and allowed to permeabilize on ice for 10 min. The membrane was then washed twice and centrifuged at 4°C, 500 g for 5 min to collect the precipitate.

(5) Resuspend the precipitate obtained in (4) with PBS buffer to obtain a resuspension, and then add 5 μL of intracellular cytoplasmic protein ZAP70 antibody (AF647 anti-mouse/human ZAP70, Biolegend, Cat# ⁶⁹¹²⁰⁶ ) or 5 μL of intracellular nuclear transcription factor FoxP3 antibody (PE anti-mouse FOXP3, ThermoeBioscience ^TM , Cat#12-5773-82) to 100 μL of the resuspension (2×10 6 cells/ml), incubate on ice for 30 min, then wash twice, centrifuge at 4°C, 500 g for 5 min, and collect the precipitate;

(6) Flow cytometry analysis was then performed.

Using 0.1% Triton as the membrane permeabilizing agent, the labeling effects of different concentrations of PFA (1.5% or 4%) on the cytoplasmic protein ZAP70 and the nuclear transcription factor Foxp3 were explored.

The results are shown in Figure 2, (A) Left: Positive control commercial reagent 1; Middle: Labeling of spleen CD3+ZAP70+ using 0.1% Triton as permeabilization agent and 1.5% PFA as fixative; Right: Labeling of spleen CD3+ZAP70+ using 0.1% Triton as permeabilization agent and 4% PFA as fixative; (B) The bar graph is the signal-to-noise ratio corresponding to the flow cytometry graph, the ratio of CD3+ZAP70+ fluorescence intensity/CD3+ZAP70- fluorescence intensity, ns, no statistical difference vs Commercial buffer 1group; (C) Left: positive control commercial reagent 2; Middle: labeling of spleen CD3+FoxP3+ using 0.1% Triton as permeabilization agent and 1.5% PFA as fixative, Right: labeling of spleen CD3+FoxP3+ using 0.1% Triton as permeabilization agent and 4% PFA as fixative; (D) The bar graph shows the signal-to-noise ratio of the flow cytometry graph, the ratio of CD3+FoxP3+ fluorescence intensity/CD3+FoxP3- fluorescence intensity, **P<0.01vs Commercial buffer 2group; It can be seen that using commercial reagent 1 (labeling cytoplasmic proteins) and commercial reagent 2 (labeling nuclear transcription factors) as comparisons, different concentrations of PFA fixation for 5 minutes and 0.1% Triton permeabilization on ice for 10 minutes can both label intracellular CD3 ⁺ ZAP70 ⁺ and CD3 ⁺ FoxP3 ⁺ cell populations. Among them, 4% PFA compared with 1.5% PFA, the intracellular CD3 ⁺ ZAP70 ⁺ cell population was more obvious and had a higher signal-to-noise ratio ( Figure 2B ).

The above results prove that 0.1% Triton as a membrane permeabilizing agent and 4% PFA as a fixative can simultaneously detect cell membrane proteins such as CD3, cytoplasmic proteins such as ZAP70, and nuclear transcription factors such as FoxP3.

2. Intracellular phosphorylated protein detection

Next, we studied the labeling of intracellular phosphorylated p38 protein based on 4% PFA as a fixative and 0.1% Triton as a permeabilizer. We also used 10 μM Anisomycin to stimulate phosphorylation of spleen cells.

The specific method for detecting the phosphorylation expression level of mouse spleen cells by flow cytometry is as follows:

(1) Preparation of mouse spleen single cell suspension: same as the above method.

(2) Add 100 μM Anisomycin (Target Mol#T6758-1 mL) to the single-cell suspension (2×10 ⁶ cells/ml) obtained in (1) above and stimulate the cells for 5 minutes; collect the cells by centrifugation.

The group without Anisomycin was used as the untreated control.

(3) Resuspend the cells collected in (2) with 100 μL of pre-cooled PBS to obtain 100 μL of resuspension, add 2 μL of membrane surface protein antibody CD11b (APC anti-mouse/human CD11b, Biolegend, Cat#101212) to the suspension, incubate on ice for 30 min, then wash with PBS; collect the precipitate by centrifugation;

(4) Resuspend the cell pellet collected in (3) above in 1 ml of 4% PFA solution, fix at room temperature for 5 min, then wash twice, centrifuge at 4°C, 500 g for 5 min, and collect the pellet;

(5) Resuspend the cell pellet collected in (4) above with 1 ml of 0.1% Triton solution, incubate on ice for 10 min for permeabilization, then wash twice, centrifuge at 4°C, 500 g for 5 min, and collect the pellet;

(6) Resuspend the precipitate collected in (5) above with PBS to obtain 100 μL of resuspension, then add 5 μL of intracellular antibody p-P38 (PE anti-p38 MAPK Phospho (Thr180/Tyr182) Antibody, Biolegend, Cat#690203) to the suspension, incubate on ice for 30 min, then wash twice, centrifuge at 4°C, 500 g for 5 min, and collect the precipitate;

(7) Flow cytometry analysis was then performed.

The results of the labeling of phosphorylated p38 by 4% PFA fixative and 0.1% Triton permeabilizer are shown in Figure 3, A) Left: spleen cells in the untreated group (not stimulated with Anisomycin); Right: spleen cells in the 10μM Anisomycin stimulated group; B) The flow cytometry histogram shows the superposition of the phosphorylated p38 fluorescence intensity in the CD11b positive group in the untreated group and the stimulated group; It can be seen that with untreated cells as negative controls, the fixation and permeabilization method with 4% PFA as a fixative and 0.1% Triton as a permeabilizer can specifically detect the expression of phosphorylated P38 protein in the cell group with high expression of CD11b. Since intracellular phosphorylation is dynamic, 4% PFA can fix the cell state, which is conducive to the detection of phosphorylated proteins.

In summary, a fixation and permeabilization scheme using 4% PFA as the fixative and 0.1% Triton as the permeabilization agent was determined, which can have a good antibody binding effect on membrane surface proteins, cytoplasmic proteins, intracellular phosphorylated proteins and nuclear transcription factors.

Example 2: Preparation and purification of antibodies conjugated with oligonucleotide tags

To confirm the successful binding of the DNA tag to the antibody, non-reducing SDS-PAGE was performed to determine the binding efficiency of the antibody-oligo tag complex.

Antibody coupled to oligonucleotide (hereinafter referred to as antibody-oligo): Antibody-oligo is obtained by connecting oligo and antibody through a click chemistry reaction of trans-cyclooctene (TCO) as a dienophile and S-tetrazine.

In the above oligonucleotide-coupled antibody preparation, the amount of antibody and oligo added was 30 pmol of oligo for every 1 μg of antibody.

Each antibody-oligo is a conjugate obtained by connecting the target protein antibody to be detected with the oligo sequence through a click chemical reaction of trans-cyclooctene (TCO) as a dienophile and S-tetrazine. The number of oligos coupled to the antibody is 2-3, the connection efficiency reaches 100%, and there is no free oligo sequence.

The specific preparation method is as follows:

1. TCO-PEG4-Oligo labeling:

(1) Prepare the following reaction system according to a volume of 10 μL: 1 μL 10xBBS (pH 7.6: 0.5 M boric acid, 1.5 M NaCl, the balance is water, pH 7.6); 1 μL 1xBBS (10xBBS diluted with water, pH about 8.5); 1 μL DMSO; 0.5 μL 100 mM TCO-PEG4-NHS (Click Chemistry Tools, Cat# A137-25) and 6.96 μL imino-modified oligo sequence (total amount of 5 nmol, synthesized by BGI);

The oligo sequence is composed of the following from 3' to 5': a poly A terminal structure (16 A's) that can be captured by the magnetic beads with oligo-dT, a barcode sequence for distinguishing antibodies (composed of 36 specific bases, satisfying the Hamming distance), a unique molecular identifier UMI (composed of 12 random bases (A, T, C, G)) and a specific sequence that can be used for PCR amplification;

The specific sequence of the above oligo sequence is as follows:

5’-CAGACGTGGTGCTCTTCCGATC*TNNNNNNNNNNNN[ANTIBODYBARCODE36]AAAAAAAAAAAAAAA*A*A-3’

In the embodiment of the present invention, the barcode sequence [ANTIBODYBARCODE36] for distinguishing antibodies is as follows: barcode of Homemade CD3-oligo: GCGTTGAAGGATATCGTGTAGACTGTCGTTAATGCC (sequence 1) barcode of Homemade CD4-oligo: CACAAGGGTCGTAATATAGGTAACAGTAGTAGGCCG (sequence 2) barcode of Homemade-p-P38-oligo: GTGTGTAGTCGCCAGTAGGTAGTTGTGTCGTAATGT (sequence 3)

Barcode of Homemade-GFP-oligo: GTGTGTAGTCGCCAGTAGGTAGTTGTGTCGTAATGT (sequence 4)

(2) After incubating the system obtained in (1) at room temperature for 15 min, 0.5 μL of 100 mM TCO-PEG4-NHS was added and incubated again at room temperature for 15 min;

(3) Add 0.2 μL of 1 M glycine solution (prepared by dissolving glycine powder in double distilled water, pH 8.5) and incubate at room temperature for 5 min to terminate all NHS groups;

(4) Using Bio-Rad Micro Bio-Spin P-6 columns, centrifuge at 1,000 g for 2 min to remove the storage solution (Tris), then add the sample to the middle of the desalting column and centrifuge at 1,000 g for 4 min to obtain a TCO-PEG4-Oligo solution (solvent: Tris);

(5) Determine the final concentration using NanoDrop and calculate according to final concentration/MW x 10 ⁶ .

2. mTz-PEG4-Antibody labeling:

(1) Wet an ultrafiltration tube that matches the size of the antibody to be concentrated with 400 μL 1x BBS;

(2) Add 50 μg of pure antibody (the antibody is not modified with fluorescent groups, and the amount used can be adjusted proportionally according to the needs) to the ultrafiltration tube, centrifuge at 14,000g for 5 minutes to concentrate the antibody (the time can be extended);

(3) Discard the filtrate, then add 400 μL 1x BBS to the ultrafiltration tube and centrifuge at 14,000 g for 5 minutes to concentrate the antibody. This process can be repeated 3-5 times (the time can be extended).

(4) Invert the ultrafiltration tube and centrifuge at 1000 g for 2 minutes to recover the antibody;

(5) Adjust the final volume of the antibody to 45 μL with 1xBBS to obtain an antibody solution;

(6) Dilute 20 mM mTz-PEG4-NHS (Click Chemistry Tools, Cat#1069-25) to 2 mM using DMSO to obtain a diluted 2 mM mTz-PEG4-NHS solution;

(7) Add 5 μL of diluted 2 mM mTz-PEG4-NHS solution and 45 μL of the antibody solution obtained in (5) above and incubate at room temperature for 30 minutes; try to ensure that the final concentration of the antibody is 1 μg/μL;

(8) Add 1 μL of 1 M glycine solution (pH 8.5) to terminate the unreacted NHS groups;

(9) Desalting the solution obtained in (8) using an ultrafiltration tube of appropriate size (repeat the above steps (2), (3), and (4));

(10) The final volume was adjusted to 50 μL with 1x BBS to obtain an mTz-PEG4-Antibody solution (solvent: 1x BBS).

3. Oligo-antibody binding

(1) Add 30 pmol of TCO-PEG4-oligo per 1 μg of mTz-PEG4-antibody. Thoroughly mix the TCO-PEG4-Oligo solution obtained in 1 and the mTz-PEG4-Antibody solution obtained in 2.

Specifically, 50 μg of mTz-PEG4-antibody was mixed with 1,500 pmol of oligonucleotide.

(2) Room temperature or 4 degrees overnight to ensure full binding;

(3) Terminate the reaction by adding 10 mM TCO-PEG4-gly (mix 10 μL 100 mM TCO-PEG4-NHS and 2 μL 1 M glycine pH 8.5, 88 μL ultrapure water, incubate at room temperature for 1 hour to terminate all NHS groups, and place at -20°C) to obtain an antibody-oligo solution.

Taking the CD3 antibody (Biolegend, Cat#155602) and the oligo sequence of 5’-CAGACGTGTGCTCTTCCGATC*TNNNNNNNNNNNNGCGTTGAAGGATATCGTGTAGACTGTCGTTAATGCCAAAAAAAAAAAAAA*A*A-3 (N is any base of A, T, C, G) as an example, the CD3 antibody-oligo prepared according to the above method was subjected to non-reducing SDS-PAGE detection with approximately 2 μg of antibody-oligo (approximately 6 μL of antibody-oligo solution) to determine the antibody binding efficiency.

The results are shown in Figure 4A, 1: Marker; 2: unmodified CD3 antibody; 3: CD3 antibody-oligo coupled with oligonucleotide sequence; it can be seen that the bound antibody CD3 antibody-oligo shows a higher molecular weight than the original antibody CD3, indicating that the antibody-oligo tag connection efficiency meets the standard.

4. Antibody-oligo salting out

The antibody-oligo solution prepared in step 3 was purified by saturated ammonium sulfate salting-out.

The details are as follows:

(1) An appropriate amount of antibody-oligo can be mixed (according to experience, if the antibody concentration is too low, there will be no subsequent precipitation);

(2) Add an equal volume of saturated ammonium sulfate solution (pH 7.1, Beijing Regen Biotechnology Co., Ltd., Cat#PP0089) and incubate on ice for 15 min;

(3) Centrifugation at full speed for 15 minutes;

(4) Resuspend the pellet in 60 μL PBS and repeat 1-2 times;

(5) Resuspend in 200 μL PBS;

(6) Wet an ultrafiltration tube of appropriate size with 200 μL 1x BBS;

(7) adding antibodies to the ultrafiltration tube for desalting ultrafiltration;

(8) Recover the antibody to obtain purified CD3 antibody-oligo, adjust the antibody-oligo concentration to 0.5 μg/μL with PBS, and add 1 μg/μL BSA (final concentration) and 0.06% sodium azide in a final volume to obtain an antibody-oligo solution with a concentration of approximately 0.5 μg/μL (solute: antibody-oligo, solvent: PBS).

Run the gel using 10% vertical electrophoresis to determine whether all unbound sequences have been removed.

The results are shown in Figure 4B, 1: Marker; 2: oligo sequence; 3: unpurified CD3-oligo antibody; 4: purified CD3-oligo antibody. Vertical electrophoresis showed that the antibody-oligo purification efficiency reached the standard.

5. Antibody-oligo antigen binding ability detection

According to the method of Example 1, a single cell suspension of C57/BL-6 mouse spleen was prepared, and 4 μL (a total of 1 μg) of the purified CD3-oligo antibody prepared in the above 4 and a commercial fluorescent protein CD3 antibody (FITC anti-mouse CD3ε, Biolegend, Cat# ¹⁰⁰³⁰⁶ ) were added to the 100 μL cell suspension (cell number was 2×10 6/ml), targeting two epitopes of CD3 protein on the surface of CD3-T cells. Incubate on ice for 30 minutes, then wash with PBS and centrifuge at 500g for 5 minutes. The cell pellet was then resuspended with 100 μL PBS, 1.25 μL of secondary antibody PE Goat anti-rat IgG (Biolegend Cat#405406) was added, incubated on ice for 20 minutes, washed with PBS, and centrifuged at 500g for 5 minutes. Flow cytometry analysis was then performed.

At the same time, a single cell suspension of spleen from athymic mice (no CD3-T cells, BALB/c Nude Mice, strain code 401, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) was prepared and the same operation was performed as a negative control.

The specific results of antibody-oligo recognition of antigens are shown in Figure 4C. Right: spleen single cell suspension of C57BL/6 mice was simultaneously labeled with CD3-FITC and CD3-oligo, indicating that CD3-oligo still has the specificity to recognize antigens; Left: athymic nude mice, the same operation was performed; it can be seen that the fluorescence intensity of the commercial flow cytometry antibody is consistent with that of the homemade CD3 antibody-oligo, and it cannot be detected in nude mice without thymic CD3 T cells, indicating that the antibody coupled with the oligonucleotide tag still has the ability to bind to the antigen and is not non-specific.

Example 3: Single-cell multi-omics INT-seq detection

1. Single-cell multi-omics INT-seq technical process

(1) preparing a single cell suspension (prepared according to the method of (1) in Example 1) and an antibody-oligo that binds to the target protein to be detected (prepared according to the method of Example 2);

(2) SSB incubation with antibody-oligo

E. coli single-stranded DNA binding protein (EcoSSB) was incubated with antibody-oligo binding to the target protein to be detected (25 μL system: 10X NEB 4 buffer (NEB Cat#B7004S) 2.5 μL; antibody-oligo titration amount; EcoSSB (Promega Cat#M3011) used in an amount of 10 times the total amount of antibody-oligo (e.g., if the antibody-oligo used is 0.3 μg, then the amount of EcoSSB used is 3 μg); the rest was filled with H ₂ O) at 37 degrees for 30 minutes to obtain antibody-oligo-EcoSSB;

The target protein may be a membrane surface protein, a cytoplasmic protein, an intracellular phosphorylated protein and/or a nuclear transcription factor of a single cell.

The antibody-oligo of different target proteins to be detected is distinguished by barcode sequence.

(3) Resuspend the cell pellet in 100 μL of blocking solution (1% BSA, 1:100 Fc (BioLegend Cat#156604), the remainder is RNase-free PBS (Beijing Regen Biotechnology Co., Ltd., Cat#NH0012)) and incubate on ice for 15 min to obtain a resuspension solution;

(4) Antibody labeling of membrane surface proteins

To the resuspension obtained in (3) above (no more than 2 x 10 ⁶ cells), add one or more membrane surface protein antibodies obtained in (2) above - oligo-EcoSSB and 20 μL of mixed sample label (BD Human Single-Cell Multiplexing Kit, Cat#633781; BD Mouse Immune Single-Cell Multiplexing Kit; Cat#633793) according to the titrated antibody usage, label the antibody in blocking solution, the total volume is 200 μL, and incubate at 4°C for 30 minutes;

(5) Add 1 mL of RNase-free PBS (containing 1% BSA) and centrifuge at 500 g for 5 min at 4°C. Repeat twice and collect the precipitate.

(6) Add 1 mL of 4% PFA fixative (purchased from Biosharp Cat#BL539A) to resuspend the cells, and add 1 μL of 40 U/mL RNase inhibitor (Roche Cat#3335399001) to resuspend the precipitate treated in (5). After fixing at room temperature for 5 minutes, add 143 μL of 1 M Glycine solution (final concentration of 0.125 M) to quench the reaction, and then centrifuge at 500 g for 5 minutes to collect the precipitate;

(7) Resuspend the precipitated cells treated in (6) with 1 mL of permeabilization solution 0.1% Triton X-100 (volume percentage, containing 1 μL 40 U/mL RNase inhibitor, the remainder is RNase-free PBS) and incubate on ice for 10 min;

(8) Centrifuge at 600 g for 5 min at 4°C, repeat twice, and collect the precipitate;

(9) Add 100 μL of blocking solution (1% BSA, 0.05% dextran amine, 1 U/mL RNase inhibitor, and the remainder is RNase-free PBS) to resuspend the precipitated cells, add one or more cytoplasmic protein antibodies-oligo-EcoSSB, one or more phosphorylated protein antibodies-oligo-EcoSSB and/or one or more nuclear transcription factor antibodies-oligo-EcoSSB according to the titrated antibody usage, and incubate on ice for 30 minutes for labeling;

(10) Add 1 mL of cleaning solution (0.02% Tween-20 solution prepared with RNase-free PBS), incubate for 5 min, then centrifuge at 4°C, 600 g for 5 min, discard the supernatant, repeat twice, and collect the precipitate;

(11) Add PBS to resuspend the precipitated cells to obtain a labeled single cell suspension;

Take an appropriate amount of labeled single cell suspension (the total amount should not exceed 50,000 cells) with a cell counter for subsequent single cell operations;

(12) A high-throughput single-cell operation platform based on a microplate is selected. The present invention takes the BD Rhapsody platform as an example, and the details are as follows:

a. Chip activation:

To process the BD Rhapsody Cartridge (BD Cat#400000847), place the waste collection tube and 5 ml low-absorption centrifuge tube in the drawer of the BD Rhapsody instrument; place the BD Rhapsody Cartridge on the instrument and process the Cartridge according to the following steps: aspirate 100% anhydrous ethanol, Prime/Treat mode; aspirate air, Prime/Treat mode; aspirate room temperature Cartridge buffer 1, Prime/Treat mode, incubate for 1 min; aspirate air, Prime/Treat mode; aspirate room temperature Cartridge buffer 1, Prime/Treat mode, incubate for 10 min; aspirate air, Prime/Treat mode; aspirate room temperature Cartridge buffer 2, Prime/Treat mode. The chip is now activated and can be left at room temperature for less than 4 hours.

b. Load cells: Adjust the P1200M pipette to the Prime/Treat mode, and aspirate 700 μL of air into the cartridge. Adjust the P1200M pipette to the Cell Load mode, and aspirate 575 μL of the fixed permeabilized single cell suspension obtained in step (11) (the total amount does not exceed 50,000 cells), so as to ensure that one single cell is captured in each well and reduce the double cell rate;

c. Loading magnetic beads: Adjust the P1200M pipette to the bead load mode, and immediately use the adjusted 1mL pipette to insert the low-adsorption pipette tip, and gently and repeatedly blow the prepared oligo-dT magnetic bead suspension (BD Rhapsody Cartridge Reagent Kit, Cat#633731) about 10 times until the magnetic beads are evenly suspended in the pre-cooled Sample Buffer (BD Rhapsody Cartridge Reagent Kit, Cat#633731). Place at 25℃ for 3 minutes. Ensure that each well corresponds to one type of magnetic bead.

d. Inhale air, Wash mode, process the Cartridge; inhale 700 μL of pre-cooled Sample Buffer to process the chip; repeat twice.

e. Lysis of cells:

Add 100 μL of lysis buffer containing proteinase K to each microwell of the chip after treatment in step d above;

The above-mentioned lysis buffer containing proteinase K is prepared by dissolving proteinase K (Qiagen Cat#19131, final concentration of 75mAU/mL) in lysis buffer (Tris-Hcl (PH=7.4) 50mM; NaCl 75mM; EDTA 6.25mM; SDS2%), that is, 100μL of proteinase K is added to 700μL of lysis buffer to obtain a lysis buffer containing proteinase K.

Slide the left slider to the "LYSIS" position; adjust the P1200M pipette to the Lysis mode, and aspirate 550μL of lysis buffer containing proteinase K; let it settle for about 25 seconds (to allow the buffer to fully settle), adjust the P1200M pipette to the Prime treat mode, aspirate 700μL of air into the cartridge, and use the Prime treat mode to aspirate 700μL of vaporlock (mineral oil, Qiagen Cat#981611), evenly cover each well, cover the well plate, and let it stand at room temperature for 5 minutes (cell lysis).

f. Recover the captured magnetic beads:

Place a 5mL low adsorption tube (Eppendorf, Cat. No. 30108310) in the instrument drawer and ensure that the P5000M pipette is set to the Retrieval mode. Slide the front slider to the "BEADS" position; slide the left slider to the "RETRIEVAL" position and time for 30 seconds. During the timing period, use the P5000M pipette to aspirate 5000ul of anhydrous ethanol. After the 30s timing is over, slide the front slider to the "0" position, immediately inject anhydrous ethanol, and recover the magnetic beads. The recovered magnetic beads capture cell mRNA and antibody-oligo linked to the target protein.

Slide the front slider to the "OPEN" position, take out the 5mL low-adsorption tube, and immediately place it on a 1.5mL magnetic stand for 1 minute to collect the captured product (antibodies containing mRNA and target protein-oligo), and wash the magnetic beads immediately after completion;

g. Construction of mRNA full transcriptome library and antibody library

The capture products obtained in step f (antibody-oligo containing mRNA and target protein) were used to construct mRNA full transcriptome library and antibody library respectively using the BD Rhapsody library construction process.

The primers used for PCR amplification in constructing the library correspond to specific sequences on the oligo that can be used for PCR amplification.

h. Sequencing

Single-cell multi-omics analysis by sequencing.

2. Single-cell multi-omics technology evaluation - transcriptome

Next, single-cell transcriptome was used to evaluate the de-crosslinking of fixed samples by the above-mentioned single-cell multi-omics technology process, and to evaluate the detection efficiency of the single-cell system for RNA in fixed samples. The experimental samples selected were HEK-293T cell line and spleen primary cells (a mouse spleen single cell isolated in Example 1).

Experimental group (denoted as INT-seq): The single-cell multi-omics technology process described above was followed for fixation and permeabilization and then the experiment was performed on the machine, and steps 2), 4), and 9) were omitted;

The two types of cells were divided into two experimental groups, and were marked with mixed sample labels respectively.

Positive control (recorded as unfixed): The single cell suspension is not fixed and permeabilized, and steps 11) and 12) of the above-mentioned single cell multi-omics technology process are directly used for the experiment.

The sequencing results of the mRNA whole transcriptome library are shown in Figure 5, where A is the number of genes (median 6,198 genes/cell) and UMI (median 27,021 counts/cell) in the unfixed HEK-293T positive control group and the number of genes (median 6,053 genes/cell) and UMI (median 28,159 counts/cell) in the fixed permeabilized HEK-293T experimental group; B is the number of genes (median 1,253 genes/cell) and UMI (median 2,428 counts/cell) in the unfixed spleen cell positive control group and the number of genes (median 1,112.5 genes/cell) and UMI (2,003 counts/cell) in the fixed permeabilized spleen cell experimental group. Therefore, at the cell line level, the single-cell transcriptome assessment of the cell line after fixed permeabilization retains 98% of the gene expression level compared to fresh samples. At the primary cell level, spleen cells that had been fixed and permeabilized retained 88% of gene expression levels compared to fresh samples, which is better than existing data.

3. Single-cell multi-omics technology evaluation - membrane surface protein detection

Single cell suspensions of spleen from C57/BL-6 mice were prepared using the same buffer as flow cytometry and labeled with homemade CD3-oligo and CD4-oligo.

The spleen single cells of C57/BL-6 mice were subjected to the experiment according to the above-mentioned single cell multi-omics technology process (denoted as INT-seq), and step 9) was omitted. Among them, the antibody-oligo in step 2) was CD3 antibody-oligo and CD4 antibody-oligo respectively; the antibody-oligo of membrane surface protein in step 4) was CD3 antibody-oligo and CD4 antibody-oligo;

In CD3 antibody-oligo, CD3 antibody (Biolegend, Cat#155602), the barcode sequence [ANTIBODYBARCODE36] for distinguishing antibodies in the oligo sequence is as follows: barcode sequence of Homemade CD3-oligo: GCGTTGAAGGATATCGTGTAGACTGTCGTTAATGCC, the titrated antibody usage is 4ul added to 100ul staining system;

In CD4 antibody-oligo, CD4 antibody (Biolegend, Cat#100506), the barcode sequence [ANTIBODYBARCODE36] for distinguishing antibodies in the oligo sequence is as follows: barcode sequence of Homemade CD4-oligo: CACAAGGGTCGTAATATAGGTAACAGTAGTAGGCCG, the titrated antibody usage is 1ul added to 100ul staining system;

A total of 15,901 cells were obtained by INT-seq, and cells with more than 500 genes were screened, and finally 14,047 cells were obtained. Cluster analysis was performed on the single-cell transcriptome sequencing data of INT-seq.

The sequencing results of the mRNA whole transcriptome library and the antibody library are shown in Figure 6, (A) UMAP clustering diagram based on INT-seq single-cell mRNA whole transcriptome data; (B) Expression distribution of characteristic gene Cd3e/Cd4 mRNA on the UMAP clustering diagram; and the expression of targeted proteins produced by INT-seq. UMAP was used for dimensionality reduction clustering. According to the expression of cell population Marker genes, ten cell populations were included, namely neutrophils, B cells, macrophages, erythrocytes, CD4 ⁺ T cells, CD8 ⁺ T cells, NK cells, cDC dendritic cells, pDC dendritic cells, and monocytes (Figure 6A). The first row is the expression level of mRNA, Cd3e is a characteristic gene of T cells; Cd4 is a characteristic gene of helper T cells. The second row is the expression of the measured antibody tag sequence. CD3-oligo is highly expressed in the T cell population, which is consistent with cell clustering and Cd3e mRNA expression. CD4-oligo was highly expressed in helper T cells, which was also consistent with cell clustering and expression of CD4 mRNA (Figure 6B). Therefore, INT-seq is accurate in detecting surface proteins.

The above results indicate that the method of the present invention can detect membrane surface proteins after single cells are fixed with 4% PFA.

4. Single-cell multi-omics technology to detect intracellular phosphorylation status

(1) HEK-293T cells and spleen primary cells were cultured normally using DMEM basic medium (containing 10% serum and 1% penicillin-streptomycin);

(2) When the cell density reached 60%-80%, the two groups of cells were cultured in serum-free medium and starved for 18 h;

(3) One group of cells was not stimulated (untreated group), and the other group of cells was stimulated with 10 μM Anisomycin (Target Mol #T6758-1 mL) for 5 min (stimulated group);

(4) directly recovering the two groups of cells, and then performing fixation and permeabilization experiments according to the above-mentioned single-cell multi-omics technology process (INT-seq), and omitting step 4), and detecting the expression of p-P38-oligo; wherein the antibody-oligo in step 9) is p-P38 antibody-oligo.

In p-P38 antibody-oligo, p-P38 antibody (Biolegend, Cat#690202), the barcode sequence [ANTIBODYBARCODE36] for distinguishing antibodies in the oligo sequence is as follows: barcode sequence of Homemade-p-P38-oligo: GTGTGTAGTCGCCAGTAGGTAGTTGTGTCGTAATGT, the titrated antibody usage is 2ul added to 100ul staining system;

At the same time, the two groups of cells first used steps 1)-8) in the above-mentioned single-cell multi-omics technology process (INT-seq), and then the cells obtained in step 8) were labeled with p-P38-PE fluorescent antibody for flow cytometry analysis.

The flow cytometry results are shown in Figure 7A. In the stimulation group, HEK-293T cells were stimulated with Anisomycin for 5 minutes, which activated the downstream P38 MAPK activity and resulted in an obvious p-P38 positive group. There was no p-P38 positive group in the unstimulated group.

The INT-seq results are shown in Figure 7B . After 5 minutes of Anisomycin stimulation of HEK-293T cells, there was an obvious p-P38 positive group, while there was no obvious clustering in the unstimulated group, which was consistent with the results of flow cytometry.

Therefore, INT-seq can accurately detect the expression of intracellular phosphorylated proteins with quantitative accuracy.

5. Single-cell multi-omics technology evaluation - intracellular protein detection

Next, we evaluated whether INT-seq can detect relative quantitative gradients of intracellular protein expression levels. Since proteins in vivo are regulated by post-translational modifications, protein levels and RNA expression levels may have a low correlation, so an in vitro transfection system was used.

The RNA expression levels measured by flow cytometry gold standard and INT-seq were used as controls for the quantitative gradient of protein expression detected by INT-seq. The self-made anti-GFP-oligo modified antibody and anti-GFP-APC fluorescent antibody were labeled in a buffer containing saturated dextran sulfate through the 4% PFA fixed 0.1% Triton permeabilization operation determined above. One part of the cells was used for INT-seq single cell analysis, and the other part of the cells was used for flow cytometry analysis.

The details are as follows:

Cell transfection: The density of HEK-293T cells should reach 60%-80% on the day of transfection experiment. Use 10cm dish or 6cm dish. Prepare Tubrofcet transfection complex according to the transfection reagent (Thermo Cat#R0531): plasmid (cytoplasmic localized GFP plasmid (plasmid expressing GFPP without nls); nuclear localized GFP plasmid (nls-EGFP, prepared by Shandong Weizhen Biotechnology Co., Ltd.): serum-free medium is 2μL: 1ug: 100μL. The total volume should not exceed 1/10 of the total culture dish culture medium. Mix together and let stand for 20 minutes; aspirate the liquid in the culture dish, wash twice with PBS, and add serum-free culture medium; add the transfection complex to the culture dish; after 8 hours, change to normal serum culture medium and culture in the incubator; after 32-42 hours, obtain HEK-293T transfected with cytoplasmic GFP fluorescent protein and HEK-293T transfected with nuclear localized GFP fluorescent protein;

The results of the fluorescence microscopy showed that HEK-293T cells transfected with cytoplasmic GFP fluorescent protein and HEK-293T cells transfected with nuclear localized GFP fluorescent protein were obtained (as shown in FIGS. 8A and 8D ).

Detection: The HEK-293T cells transfected with cytoplasmic GFP fluorescent protein and the HEK-293T cells transfected with nuclear localized GFP fluorescent protein were subjected to flow cytometry and INT-seq single-cell operations, respectively.

The INT-seq single-cell operation was performed according to the single-cell multi-omics technology process (INT-seq) described above, followed by fixation and permeabilization, and step 4) was omitted; wherein the antibody-oligo in step 2) was GFP antibody-oligo; and the antibody-oligo in step 9) was GFP antibody-oligo.

In GFP antibody-oligo, GFP antibody (Biolegend Cat#338002), the barcode sequence [ANTIBODYBARCODE36] in the oligo sequence that distinguishes the antibody is as follows: barcode sequence of Homemade-GFP-oligo: GTGTGTAGTCGCCAGTAGGTAGTTGTGTCGTAATGT, the titrated antibody usage is 6ul added to 100ul staining system;

Flow cytometry used steps 1)-8) in the above-mentioned single-cell multi-omics technology process (INT-seq), omitting step (4), and then the cells obtained in step 8) were labeled with anti-GFP antibody, labeled with secondary antibody, APC Goat anti-mouse IgG (Biolegend Cat#405308) for flow analysis.

The results of flow cytometry analysis are shown in Figures 8B and 8E. Based on the autofluorescence FITC of GFP, three groups of cells were flow sorted: GFP negative (-/-); GFP medium expression (+); and GFP high expression (++), and were marked with mixed sample labels respectively (Figure 8B/E).

The INT-seq detection results are shown in Figures 8C and 8F. The single-cell results show that the method of the present invention can flow cytometry sort out three groups of cells: GFP negative (-/-); GFP medium expression (+); and GFP high expression (++), indicating that the method of the present invention is correct.

Claims (10)

1. A single-cell multi-omics detection method for paraformaldehyde-fixed cells, comprising the following steps: A. Use 4% PFA as fixative and 0.1% Triton as permeabilization agent to fix and permeabilize single cells. B. Lysing the fixed and permeabilized cells in a single-cell operating system, followed by library construction and sequencing to achieve single-cell multi-omics detection of paraformaldehyde-fixed cells; The lysis is performed in a high-throughput single-cell microplate system of the single-cell operating system. 2. The method according to claim 1, characterized in that: If the single-cell multi-omics detection is mRNA whole transcriptomics detection, the method includes step A and step B; If the single-cell multi-omics detection is a membrane surface protein detection, then before the step A, the following step A-1 is also included: labeling the single cell with one or more antibody-oligos that bind to the membrane surface protein to be detected to obtain a membrane surface protein labeled cell; If the single-cell multi-omics detection is a non-membrane surface protein detection, then between the steps A and B, the following step A-2 is also included: labeling the fixed permeabilized cells with one or more antibodies-oligos that bind to the non-membrane surface protein to be detected to obtain labeled cells; The non-membrane surface protein is a cytoplasmic protein, a phosphorylated protein and/or a nuclear transcription factor; If the single-cell multi-omics detection is mRNA full transcriptomics detection and membrane surface protein detection, the method includes A-1, A and B; If the single-cell multi-omics detection is mRNA whole transcriptomics detection and non-membrane surface protein detection, the method includes A, A-2 and B; If the single-cell multi-omics detection is mRNA whole transcriptomics detection, membrane surface protein detection and non-membrane surface protein detection, the method includes A-1, A, A-2 and B. 3. The method according to claim 1 or 2, characterized in that: The single-cell operating system is a BD Rhapsody single-cell operating system; The high-throughput single-cell microplate system is the BD Rhapsody system. 4. The method according to claim 3, characterized in that: The step B comprises the following steps: performing high-throughput single-cell detection on the fixed permeabilized cells in a BD Rhapsody system microplate system, and after the cells are added to the BD Rhapsody system microplate, sequentially performing the following steps: adding magnetic beads for capturing nucleic acids, cell lysis and enrichment of magnetic beads to obtain single-cell mRNA and/or all antibody-oligos connected to proteins, and then using the single-cell mRNA and/or all antibody-oligos connected to proteins to construct an mRNA full transcriptome library and/or an antibody library respectively; sequencing to achieve single-cell multi-omics detection of paraformaldehyde-fixed cells; Alternatively, one single cell and one magnetic bead are added to each microwell of the BD Rhapsody system; Alternatively, the nucleic acid is mRNA and/or all antibody-oligos from a single cell. 5. The method according to claim 4, characterized in that: The magnetic beads used for capturing nucleic acid are magnetic beads with oligo-dT, and the oligo-dT in the magnetic beads captures the antibody of protein-polyA in the oligo; Alternatively, the lysis solution used for cell lysis is a lysis buffer containing proteinase K. 6. The method according to claim 5, characterized in that: In each microwell, the concentration of proteinase K in the lysis buffer containing proteinase K is 75mAU/mL, and the added volume is 100ul; Or, in the cell lysis, the step of adding mineral oil is further included after adding the lysis buffer containing proteinase K; Alternatively, the enriched magnetic beads are recovered by anhydrous ethanol. 7. The method according to any one of claims 2 to 6, characterized in that the antibody-oligo of each protein is an antibody-oligo of a protein that binds to SSB. 8. The method according to any one of claims 2 to 7, characterized in that: In step A-2), the labeling is performed in a buffer containing saturated dextran sulfate. 9.4% paraformaldehyde was used as a fixative and 0.1% Triton was used as a permeabilizer in fixing single cells. 10. Use of step B in any one of the methods of claims 1 to 8 in detecting the multi-omics status of single cells after 4% PFA fixation; Or, 4% paraformaldehyde is used as a single cell fixative and 0.1% Triton is used as a single cell permeabilizing agent, and step B of the method according to any one of claims 1 to 8 is used in single cell multi-omics detection.