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WO2022067494A1 - Procédé de détection de transcriptome entier dans des cellules individuelles - Google Patents

Procédé de détection de transcriptome entier dans des cellules individuelles Download PDF

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Publication number
WO2022067494A1
WO2022067494A1 PCT/CN2020/118794 CN2020118794W WO2022067494A1 WO 2022067494 A1 WO2022067494 A1 WO 2022067494A1 CN 2020118794 W CN2020118794 W CN 2020118794W WO 2022067494 A1 WO2022067494 A1 WO 2022067494A1
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rna
oligo
poly
dtn6
cell
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English (en)
Inventor
Jiameng SUN
Guoxin Tang
Ji Li
Tao Li
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Singleron Nanjing Biotechnologies Ltd
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Singleron Nanjing Biotechnologies Ltd
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Priority to PCT/CN2020/118794 priority Critical patent/WO2022067494A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present disclosure is a high-throughput method for detecting a single cell full transcriptome sequence, which involves single cell analysis and cell culture, and in particular relates to the detection of single cell full transcriptome sequence.
  • Single-cell sequencing is a technology that performs amplification and sequencing of DNA [1] or RNA [2] in units of single cells, Compared with conventional sequencing, this technology can accurately adjust the resolution of gene expression from the average of the population level to the level of individual cells.
  • Massively Parallel Single Cell Sequencing based on microfluidic technology can separate a large number of cells into single cells and add molecular tags to each single cell and then jointly amplify and construct a library for sequencing [3] , avoiding manual cumbersome experimental procedure for separating single cells.
  • the most commonly used high-throughput single cell sequencing method is single cell mRNA sequencing, where the 3’ of the mRNA in each individual cell is quantitatively detected by sequencing.
  • Expression profiles of mRNA in single cells can be then used to annotate different cell types in a sample, and also to discover gene and pathway characteristics in each cells.
  • the data and insights generated by single cell mRNA sequencing greatly enrich knowledge in diverse fields such as cancer [4] , neurology [5] , and immunology [6] , and facilitate improvements in diagnosis and treatment of diseases.
  • ncRNAs Non-coding RNAs
  • lncRNAs Long non-coding RNAs form a majority of the human transcriptome and play key roles in the cellular and physiological functions, such as chromatin dynamics, gene expression, cell growth and differentiation [9] .
  • lncRNAs Changes in lncRNA expression and their mutations promote tumor occurrence and metastasis, and different lncRNAs may exhibit tumor inhibition and promotion functions [10] . Due to their tissue-specific expression characteristics and relevance in oncology, lncRNAs can be used as new biomarkers and targets for the treatment of cancer. This information is of great significance for the analysis of cell gene expression and regulation.
  • Micro RNAs are small non-coding RNAs approximately 20 to 22 nucleotides long, which play very important roles in the regulation of target genes by binding to complementary regions of mRNAs to repress their translation or regulate their degradation [11] . This regulation appears to be involved in many fundamental cellular processes, including development, differentiation, proliferation, stress response, metabolism, apoptosis and secretion [12] .
  • Other ncRNA species such as snoRNA and circle RNA, have all be implicated in various aspects of the cellular functions.
  • RNA expression analysis starts by extracting total RNA from samples and then analyze total RNA, or ribosomal RNA –depleted RNA, with sequencing, microarray, or PCR [13, 14] .
  • the expression level of ncRNAs in bulk sample is an average of that in all cell types in the sample, which can mask cell-specific ncRNA expression patterns that are functionally relevant.
  • mRNA can be regularly detected at single cell level by methods such as SMART-seq, such methods generally start with capturing mRNA molecules through their 3’ poly-A tails with an oligo-dT RT primer [15] . Most ncRNA molecules do not have poly-A tails and cannot be captured this way at single cell level.
  • SUPeR-seq is such a method that replaces commonly used oligo (dT) primers with random primers with anchor sequences, and can simultaneously capture polyA + and polyA-RNA in a 96-well plates.
  • polyA + RNA was captured through dT15 + N6 region [16]
  • polyA-RNA was captured through N6 random primers.
  • this technology is limited by the number of single cells and cannot achieve massive single-cell whole transcriptome sequencing, the number of single cells is insufficient, which is not conducive to analysis such as cell clustering.
  • RamDA-seq uses short NSRs (not-so-random primers) to capture and reverse transcribe RNAs while excluding rRNA in a 96-well plates [17] .
  • NSRs not-so-random primers
  • oligo dT and N6 random primer magnetic beads to capture poly A+ RNA and poly A-RNA
  • These magnetic beads oligo have combined with barcode and UMI sequence that can distinguish each single cell from other cells and label the transcripts, so that thousands or more of single cells can be analyzed in parallel.
  • the RNA captured by the dTN6 magnetic beads is subjected to reverse transcriptase using the magnetic beads oligo sequence as a primer to synthesis the first strand of cDNA, than add a specific primer site to the first strand cDNA , the second strand cDNA synthesis through this site, the obtained double-stranded cDNA is enriched by amplification, the product is used for library construction and sequencing.
  • This method can also be used in combination with a microfluidic system where each cells in a sample can be partitioned to individual micro-chambers. Single cells can be lyzed in the micro-chambers than the RNA were released and captured by dTN6 magnetic beads.
  • Figure 1 Schematic diagram of the present disclosure.
  • Figure 2 Schematic diagram of the embodiment of the present disclosure where RNA were captured by dTN6 magnetic beads.
  • Figure 3 shows the percentage of UMI.
  • One embodiment of the present disclosure is to use dTN6 magnetic beads to capture Poly A +RNA and Poly A -RNA. Afterwards, oligo sequence can be used as primer to reverse transcribe both mRNA and ncRNA. The resulting cDNA can be amplified by PCR if a template switching oligo is introduced during the RT process. With unique cell barcodes in conjunction with the oligo sequence, cDNA molecules from the same single cell can be labeled and a group of single cells can be processed in parallel, enabling high-throughput single cell analysis (Figure 2) .
  • GEXSCOPE Single Cell RNAseq Library Construction kit (Singleron Biotechnologies) was used to demonstrate the technical feasibility and the utility of the present disclosure in massively parallel single cell ncRNA sequencing. The experiment was conducted according to manufacturer’s instructions with modifications described below.
  • single cell suspension of CCRF cells was loaded onto the microchip to partition single cells into individual wells on the chip.
  • Four samples were prepared: one was processed with standard GEXSCOPE protocol for single cell mRNA sequencing ( “control” ) , another was processed with modified protocol to get ncRNA reads ( “experiment” ) .
  • Cell barcoding magnetic beads were then loaded into the microchip of “control” group and washed.
  • Cell barcoding and dTN6 magnetic beads were then loaded into the microchip of “experiment” group and washed. Both magnetic beads contain oligos with a unique cell barcode sequence combined with oligo on the surface.
  • Each oligo on the bead also has a unique molecule index sequence (UMI) ; the number of UMIs detected in the sequence can be used to accurately quantify different RNA molecules. Only one bead can fall into each well on the microchip based on the diameters of the beads and well (about 30um and 40um, respectively) .
  • UMI unique molecule index sequence
  • RNAseq library was sequenced on Illumina NovaSeq with PE150 mode and analyzed with scopeTools bioinformatics workflow (Singleron Biotechnologies) .
  • the percentage of the UMIs corresponding to ncRNA in total UMIs increased about 100%, from 1.17%to 2.38 %.
  • the significantly increased percentage of ncRNA UMIs proves the principle of the present disclosure.
  • the percentage of rRNA UMIs remains relatively at low level .

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé, basé sur la puce microfluidique développée par Singleron Independent pour séparer des cellules uniques, utilisant des billes magnétiques oligo dT et N6 pour capturer l'ARN poly A+ et l'ARN poly A, et l'ARN capturé est ensuite transcrit de manière inverse en ADNc. L'ADNc résultant peut alors être amplifié et analysé. Le séquençage du transcriptome entier au niveau des cellules individuelles massivement parallèles fournit des données plus utiles pour l'analyse de profils d'expression cellulaire et de mécanismes de régulation.
PCT/CN2020/118794 2020-09-29 2020-09-29 Procédé de détection de transcriptome entier dans des cellules individuelles Ceased WO2022067494A1 (fr)

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PCT/CN2020/118794 WO2022067494A1 (fr) 2020-09-29 2020-09-29 Procédé de détection de transcriptome entier dans des cellules individuelles

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023236121A1 (fr) * 2022-06-08 2023-12-14 深圳华大生命科学研究院 Procédé de détection d'une cellule rare, appareil et utilisation de celui-ci
CN119161969A (zh) * 2024-08-08 2024-12-20 北京化工大学 一种用于组织细胞核空间转录组测序的微流控芯片

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WO2015168161A2 (fr) * 2014-04-29 2015-11-05 Illumina, Inc. Analyse de l'expression de gènes de cellules isolées multiplexées par commutation de matrice et fragmentation et étiquetage (tagmentation)
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WO2018064640A1 (fr) * 2016-10-01 2018-04-05 Berkeley Lights, Inc. Compositions de code-barres d'adn et procédés d'identification in situ dans un dispositif microfluidique
CN108103055A (zh) * 2018-01-09 2018-06-01 上海亿康医学检验所有限公司 一种单细胞rna逆转录与文库构建的方法
CN108949909A (zh) * 2018-07-17 2018-12-07 厦门生命互联科技有限公司 一种用于基因检测的血小板核酸文库构建方法和试剂盒
CN109971843A (zh) * 2017-12-27 2019-07-05 复旦大学泰州健康科学研究院 一种单细胞转录组的测序方法
CN110684829A (zh) * 2018-07-05 2020-01-14 深圳华大智造科技有限公司 一种高通量的单细胞转录组测序方法和试剂盒

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015031691A1 (fr) * 2013-08-28 2015-03-05 Cellular Research, Inc. Analyse massivement parallèle de cellules uniques
WO2015168161A2 (fr) * 2014-04-29 2015-11-05 Illumina, Inc. Analyse de l'expression de gènes de cellules isolées multiplexées par commutation de matrice et fragmentation et étiquetage (tagmentation)
WO2016040476A1 (fr) * 2014-09-09 2016-03-17 The Broad Institute, Inc. Procédé à base de gouttelettes et appareil pour l'analyse composite d'acide nucléique de cellules uniques
WO2017040306A1 (fr) * 2015-08-28 2017-03-09 Illumina, Inc. Analyse de séquences d'acides nucléiques provenant de cellules isolées
WO2018064640A1 (fr) * 2016-10-01 2018-04-05 Berkeley Lights, Inc. Compositions de code-barres d'adn et procédés d'identification in situ dans un dispositif microfluidique
CN109971843A (zh) * 2017-12-27 2019-07-05 复旦大学泰州健康科学研究院 一种单细胞转录组的测序方法
CN108103055A (zh) * 2018-01-09 2018-06-01 上海亿康医学检验所有限公司 一种单细胞rna逆转录与文库构建的方法
CN110684829A (zh) * 2018-07-05 2020-01-14 深圳华大智造科技有限公司 一种高通量的单细胞转录组测序方法和试剂盒
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023236121A1 (fr) * 2022-06-08 2023-12-14 深圳华大生命科学研究院 Procédé de détection d'une cellule rare, appareil et utilisation de celui-ci
CN119161969A (zh) * 2024-08-08 2024-12-20 北京化工大学 一种用于组织细胞核空间转录组测序的微流控芯片

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