WO2018149091A1 - Method for constructing circular rna high-throughput sequencing library and kit thereof - Google Patents

Abstract

Disclosed are a method for constructing a circular RNA high-throughput sequencing library and a kit thereof. The construction method sequentially comprises the steps of: (S1) extracting total RNA from a sample; (S2) removing DNA from the sample; (S3) detecting and evaluating the quality of the total RNA in the sample, and determining that the RNA quality meets requirements; (S4) removing rRNA; (S5) removing linear RNA; and (S6) constructing the circular RNA library for high-throughput sequencing. The method for constructing the circular RNA high-throughput sequencing library is high in efficiency and stable, and has a low residual ratio of the rRNA, is high in circular RNA detection efficiency, good in data repeatability, and has a high verification success rate for the sequencing results, and is especially applicable to an FFPE tissue and other samples with a poor RNA quality.

Description

Method for constructing circular RNA high-throughput sequencing library and kit thereof Technical field

The invention belongs to the field of molecular biology, and particularly relates to a method for constructing a high-throughput sequencing library of circular RNA and a kit thereof.

Background technique

A circular RNA (circuRNA) is a new member of the RNA family that differs from traditional linear RNA in that it does not have a 5' end cap and a 3' end poly(A) tail and forms a ring structure with a covalent bond. Encode RNA molecules. As early as 1980, circular RNA has been discovered, but for a long time, due to the limitations of research technology, circular RNA is considered as a by-product of erroneous variable shear, which belongs to nature. Very rare phenomena, and even caused by genetic accidents or experimental human factors, have not attracted the attention of the academic community. With the development of deep RNA sequencing and large-scale bioinformatics, researchers have discovered that a large number of circular RNA molecules exist in living organisms. In 2012, Salzman et al. discovered that circular RNA is widely present in the human transcriptome by high-throughput sequencing, which has attracted interest in circular RNA research. In 2013, Hansen et al. found that CDR1as/CiRS-7 can regulate gene expression as a miR-7sponge, confirming the important role of circular RNA in humans. Recent studies have shown that circular RNA has a closed loop structure, mainly produced by atypical variable shear processing, widely existed in various biological cells, has structural stability, is difficult to be degraded by RNase, has high expression abundance, and is conserved between species. Good sex, expression has the characteristics of tissue and time and space specificity. Studies have shown that circular RNA is associated with diseases such as neurodevelopment, atherosclerosis, myotonic dystrophy, cancer, etc., and the presence of circular RNA in human saliva and blood indicates that the circular RNA can be It is stable in clinical specimens such as blood, urine and ascites. These characteristics make the circular RNA have broad prospects in the development and application of new disease diagnosis and treatment methods.

CN 104388548 A discloses a method for high-throughput circular RNA sequencing comprising: artificially synthesizing exogenous linear ERCC-0004-RNA and exogenous circular ERCC-0013-RNA; exogenous circular ERCC-0013-RNA Quality control; mixing exogenous linear ERCC-0004-RNA and exogenous cyclic ERCC-0013-RNA in equimolar ratio to obtain mixed exogenous RNA; adding mixed exogenous RNA to total RNA of the sample to be sequenced a first mixture; removing the ribosomal RNA in the first mixture to obtain a second mixture; performing a 3' end biotin labeling on the second mixture, and removing the lasso RNA and linear RNA labeled with biotin to obtain a third mixture; Removing the linear RNA from the third mixture to obtain a fourth mixture The standard mixture was subjected to standard high-throughput transcriptome sequencing and biological analysis of the sequencing data. This method is complicated and requires the synthesis of exogenous RNA in advance.

Since circular RNA cannot be directly isolated, there is no standard method for constructing circular RNA libraries in the literature, and most studies use methods for removing rRNA and removing linear RNA, and then constructing a library for high-throughput sequencing. There are also a small number of studies that directly build libraries after removing rRNA, or directly build libraries after removing linear RNA, and a very small part of the study is to use the method of removing PolyA+RNA. These methods have disadvantages such as incomplete removal of rRNA and poor data repeatability. Therefore, how to efficiently and stably construct a library for high-throughput sequencing of circular RNA is still a technical problem to be solved.

In addition, there is no specific circular RNA library kit on the market, and researchers need to purchase RNA extraction, rRNA removal, linear RNA removal, library construction, etc. from QIAGEN, Illumina, Backman, NEB, Life Technology and other companies. Products, and then explore the appropriate conditions for building a library. Such a combination of multiple reagents is expensive on the one hand, and researchers on the other hand need higher experimental skills to spend a long time exploring the best experimental conditions, which severely limits the progress of circular RNA research, so it is necessary Develop a dedicated kit for efficient and stable construction of high-throughput sequencing libraries for circular RNA.

Summary of the invention

In view of the above problems, the inventors of the present invention have repeatedly studied and analyzed circular RNA sequencing, and developed a method for efficiently and stably constructing a high-throughput sequencing library of circular RNA, which has a low rRNA residual ratio and data repeatability. Good, and the method is especially suitable for samples with poor RNA quality such as FFPE tissue.

In order to achieve the above object, the present invention provides a method for constructing a circular RNA high-throughput sequencing library, which in turn comprises the following steps:

(S1) extracting total RNA in the sample;

(S2) removing DNA from the sample;

(S3) Detecting and assessing the total RNA quality in the sample to determine that the RNA quality meets the requirements;

(S4) removing rRNA;

(S5) removing linear RNA;

(S6) Construction of a circular RNA library for high throughput sequencing.

Preferably, in the method of constructing the circular RNA high-throughput sequencing library, the total RNA extracted in the sample in the step (S1) is performed by the Trizol method, and the method is particularly suitable for tissue and cell samples.

Preferably, in the method of constructing the circular RNA high-throughput sequencing library, the removal of DNA in the sample in the step (S2) is carried out by DNase I digestion.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, detecting the total RNA quality in the sample in the step (S3) comprises the following steps:

(S31) Using 1% agarose gel electrophoresis to detect the degree of RNA degradation and whether there is contamination;

(S32) detecting the purity of the RNA by using an ultraviolet spectrophotometer, that is, an OD260/280 ratio;

(S33) Detection of RNA integrity.

More preferably, in the construction method of the circular RNA high-throughput sequencing library, the detection of RNA integrity in the step (S33) is performed using an Agilent 2100 bioanalyzer.

In the present invention, OD (optical density) means the optical density absorbed by the substance to be detected, and OD260/280 ratio means the ratio of the absorbance luminosity at wavelengths of 260 nm and 280 nm for judging the purity of the RNA. Theoretically, in the case of pure RNA, the OD260/280 ratio is 2, where OD260 represents the absorbance of the nucleic acid and OD280 represents the absorbance of the protein.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the removal of the rRNA in the step (S4) is performed by the RNase H digestion method.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S4) of removing the rRNA sequentially comprises the following steps:

(S41) mixing a mass of a DNA probe complementary to the rRNA sequence to form a DNA probe library;

(S42) mixing and obtaining hybridization of the obtained DNA probe library with RNA which has been tested and evaluated for total RNA quality in the sample to determine the quality of the RNA, to form a DNA-RNA hybrid;

(S43) digesting the obtained DNA-RNA hybrid with RNase H;

(S44) digesting the DNA probe with DNase I to obtain rRNA depleted RNA;

(S45) The concentration of rRNA depleted RNA was measured by a fluorometer.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the weight ratio of the DNA probe library to the RNA used in the step (S42) is (1-2): 1, preferably 1:1. .

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the conditions for hybridization in the step (S42) are: hybridization at a temperature of 95 ° C for 2 minutes, and then at 0.1 ° C / s. The speed slowly reduces the temperature to 45 °C.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the RNase is used in the step (S43) The DNA-RNA hybrid obtained by H digestion was subjected to digestion at a temperature of 37 ° C for 30 minutes.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the fluorometer used to determine the concentration of rRNA-depleted RNA in the step (S45) is performed by a Qubit fluorometer.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the linear RNA removal in the step (S5) is performed by using the RNase R digestion method, wherein the ratio of the RNase R to the RNA is (2.5- 3.5) U: 1 ug, preferably 3:1, ie, 3 active units (U) of RNase R are added per 1 microgram of RNA.

More preferably, in the method for constructing the circular RNA high-throughput sequencing library, the digestion condition of the RNase R digestion method for removing linear RNA in the step (S5) is 35-45 ° C, preferably 37 ° C. Digestion at a temperature of 20-40 minutes, preferably 30 minutes.

Preferably, in the method of constructing the circular RNA high-throughput sequencing library, the construction of the circular RNA library for high-throughput sequencing in the step (S6) is carried out by the dUTP method.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, constructing the circular RNA library for high-throughput sequencing in the step (S6) sequentially comprises the following steps:

(S61) RNA fragmentation;

(S62) First strand synthesis: using reverse transcriptase plus random primers;

(S63) second strand synthesis: digesting the RNA strand in the RNA/cDNA hybrid using RNase H, and then synthesizing the second strand by DNA polymerase I;

(S64) End repair: formation of blunt-end dsDNA using End Repair Mix and addition of a phosphate group at the 5' end;

(S65) plus A tail: use Klenow 3'-5'exo- to add A tail at the end of dsDNA;

(S66) Y-type Adapter connection: use T4DNA Ligase to connect Adapter and d-DNA with A tail;

(S67) second strand digestion and library amplification: digestion of the second strand with UNG enzyme;

(S68) Quality inspection and preparation of the index library.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the condition of RNA fragmentation in the step (S61) is fragmentation at a temperature of 90-96 ° C, preferably 94 ° C for 3-6 minutes. , preferably 5 minutes.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the peak after RNA fragmentation in the step (S61) is located at 300-350 bp.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S62) of the first strand synthesis utilizes actinomycin D to inhibit the DNA-dependent DNA polymerase activity of the reverse transcriptase to retain Directional information of the chain.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S66) Y-type Adapter The Adapter end in the junction is linked to T by a phosphorothioate linkage to prevent formation of an Adapter dimer. Further preferably, the purification of the ligation product is purified once with 1×beads, and the fragment size is selected by 0.7×/0.2×beads to select a suitable one. Fragment of the size.

Preferably, in the method for constructing the circular RNA high-throughput sequencing library, the step (S68) of the quality inspection and preparation of the indexing library sequentially comprises the following steps:

(S681) detecting the concentration of the library;

(S682) using Agilent 2100 to detect library quality;

(S683) According to the library concentration, different indexing libraries were mixed and prepared for sequencing on the machine.

Compared with the conventional method, the method for constructing the high-throughput sequencing library of the circular RNA provided by the invention is efficient and stable, the ratio of rRNA residues is low, the detection efficiency of the cyclic RNA is high, the data repeatability is good, and the success rate of the sequencing result is verified. High, especially suitable for samples with poor RNA quality such as FFPE tissue.

In addition, the present invention also provides a kit for constructing a circular RNA high-throughput sequencing library, comprising:

(1) a circular RNA enrichment reagent, DNase I, RNase H, RNaseR;

(2) species-specific DNA probes;

(3) Various enzymes required for library construction: DNA polymerase I, Large (Klenow) Fragment, T4 Polynucleotide Kinase (T4PNK), Klenow Fragment, Escherichia coli UDG, T4 DNA Ligase (Rapid), Phusion High-Fidelity DNA Polymerase ;

(4) magnetic beads, dNTP, Nuclease Free Water, dUTP, connectors, primers, etc.

Wherein the species-specific DNA probe is complementary to the rRNA sequence of the corresponding species.

The circular RNA high-throughput sequencing library construction kit provided by the invention has simple operation, optimized conditions and low cost, and is a powerful tool for circular RNA research.

Compared with the prior art, the present invention has the following advantages and benefits:

(1) Construction method of high-throughput sequencing library of circular RNA of the present invention Since a species-specific DNA probe library is established, RNaseH is used to remove rRNA bound to probe, and RNaseR is further enriched for circular RNA, so rRNA Low residual ratio, high detection efficiency of circular RNA, good data repeatability, high success rate of sequencing results, especially suitable for samples with poor RNA quality such as FFPE tissue;

(2) The kit for constructing a high-throughput sequencing library of the circular RNA of the present invention is simple, convenient, and low in cost, and is a powerful tool for research of circular RNA.

DRAWINGS

1 is a flow chart showing an embodiment of a method for constructing a circular RNA high-throughput sequencing library of the present invention;

2-1 is a result of quality inspection of a library using Agilent 2100 after completion of database construction of a circular RNA high-throughput sequencing library in the cell sample of Example 1 of the present invention;

2-2 is a result of quality inspection of a library using Agilent 2100 after completion of database construction of a circular RNA high-throughput sequencing library in a fresh liver cancer tissue sample according to Example 2 of the present invention;

2-3 is a result of quality inspection of a library using Agilent 2100 after completion of database construction of a circular RNA high-throughput sequencing library in the FFPE sample of Example 3 of the present invention;

3 is a result of QPCR verification after selecting 20 circular RNAs after sequencing of the cell sample of Example 1.

detailed description

In order to make the objects, technical solutions and advantages of the present invention more comprehensible, the present invention will be further described in detail with reference to the accompanying drawings.

Specifically, as shown in FIG. 1 , an embodiment of the present invention provides a method for constructing a high-throughput sequencing library of circular RNA, which in turn includes the following steps:

S1, extracting total RNA in the sample;

The total RNA extracted from the sample is performed using the Trizol method, which is particularly suitable for tissue and cell samples.

S2, removing DNA from the sample.

Preferably, removal of DNA from the sample is carried out using the DNase I digestion method.

S3, detecting and assessing the total RNA quality in the sample to determine the RNA quality meets the requirements.

Preferably, detecting and assessing the total RNA quality in the sample comprises the following steps:

(S31) Using 1% agarose gel electrophoresis to detect the degree of RNA degradation and whether there is contamination;

(S32) detecting the purity of the RNA by using an ultraviolet spectrophotometer, that is, an OD260/280 ratio;

(S33) Detection of RNA integrity.

The integrity of the RNA detected in the step (S33) was performed using an Agilent 2100 Bioanalyzer.

S4, rRNA is removed.

Removal of rRNA was performed using the RNase H digestion method.

The removal of rRNA by RNase H digestion involves the following steps:

(S41) mixing a mass of a DNA probe complementary to the rRNA sequence to form a DNA probe library;

(S42) mixing and obtaining hybridization of the obtained DNA probe library with RNA which has been tested and evaluated for the total RNA quality of the RNA in the sample to form a DNA-RNA hybrid;

(S43) digesting the obtained DNA-RNA hybrid with RNase H;

(S44) digesting the DNA probe with DNase I to obtain rRNA depleted RNA;

(S45) The concentration of rRNA depleted RNA was measured by a fluorometer.

The mass ratio of the DNA probe library to RNA used in the step (S42) is (1-2): 1, preferably 1:1.

The conditions for hybridization in the step (S42) were as follows: hybridization was carried out at a temperature of 95 ° C for 2 minutes, then the temperature was slowly lowered to 45 ° C at a rate of 0.1 ° C / s, and incubated at 45 ° C for 5 minutes.

The DNA-RNA hybrid obtained by digesting with RNase H in the step (S43) was subjected to digestion at a temperature of 37 ° C for 30 minutes.

The fluorometer used to determine the concentration of rRNA depleted RNA in the step (S45) was performed by a Qubit fluorometer.

The product obtained in the step (S45) was further examined by an Agilent 2100 instrument to confirm that no typical peak map of 18S, 28S rRNA was observed.

S5, removes linear RNA.

The removal of linear RNA is carried out by RNase R digestion, wherein the ratio of RNase R to RNA is (2.5-3.5) U: 1 ug, preferably 3:1, that is, adding 3 active units (U) per 1 microgram of RNA. RNase R.

The digestion conditions for the RNase R digestion method used to remove linear RNA are digestion at a temperature of 35-45 ° C, preferably 37 ° C, for 20-40 minutes, preferably 30 minutes.

S6, construction of a circular RNA library for high throughput sequencing.

Construction of a circular RNA library for high-throughput sequencing was performed using the dUTP method.

The dUTP method constructs a circular RNA library for high-throughput sequencing, which in turn includes the following steps:

(S61) RNA fragmentation;

(S62) First strand synthesis: using reverse transcriptase plus random primers, the raw materials are dNTPs (dA, dC, dG and dT each 25 mM);

(S63) second strand synthesis: the RNA strand in the RNA/cDNA hybrid is digested with RNase H, and then the second strand is synthesized by DNA polymerase I, the raw material is dUTP mix (10 mM dA, dC, dG and 20 mM dU);

(S64) End repair: formation of blunt-end dsDNA using End Repair Mix and addition of a phosphate group at the 5' end;

(S65) plus A tail: use Klenow 3'-5'exo- to add A tail at the end of dsDNA;

(S66) Y-type Adapter connection: use T4DNA Ligase to connect Adapter and d-DNA with A tail;

(S67) second strand digestion and library amplification: digestion of the second strand with UNG enzyme;

(S68) Quality inspection and preparation of the index library.

The conditions for RNA fragmentation in the step (S61) are fragmentation at a temperature of 90-96 ° C, preferably 94 ° C, for 3-6 minutes, preferably 5 minutes.

The peak after RNA fragmentation in the step (S61) is located at 300-350 bp.

The first strand synthesis of the step (S62) utilizes actinomycin D to inhibit the DNA-dependent DNA polymerase activity of the reverse transcriptase to retain the directional information of the strand.

In the step (S66), the Adapter end of the Y-type Adapter is connected with T by a phosphorothioate bond to prevent formation of an Adapter dimer. Further preferably, the purification of the ligation product is purified once with 1×beads, 0.7×/0.2×. The beads are screened for fragment size to select fragments of the appropriate size.

The step (S68) of the quality inspection and preparation of the indexing library comprises the following steps:

(S681) Qubit fluorometer detects library concentration;

(S682) using Agilent 2100 to detect library quality;

(S683) According to the library concentration, different indexing libraries were mixed and prepared for sequencing on the machine.

Compared with the conventional method, the method for constructing the high-throughput sequencing library of the circular RNA provided by the invention is efficient and stable, the ratio of rRNA residues is low, the detection efficiency of the cyclic RNA is high, the data repeatability is good, and the success rate of the sequencing result is verified. High, especially suitable for samples with poor RNA quality such as FFPE tissue.

The present invention will be elucidated with the following specific examples, but the present invention is by no means limited to these embodiments. The following examples are merely preferred embodiments of the invention, and are merely illustrative of the invention and are not to be construed as limiting the scope of the invention. It should be noted that any modifications, equivalent substitutions and improvements made within the spirit and scope of the invention are intended to be included within the scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Example 1 Construction of a High-throughput Sequencing Library of Circular RNA in Cell Samples

(S1) Extraction of total RNA from cell samples

1 × 10 ⁷ cell samples were taken, washed once with PBS at 4 ° C, then 1 mL of Trizol was added to each well of a 6-well plate, and repeatedly blown 10 times with a 1 mL tip. The sample was collected into an EP tube, 200 μL of chloroform was added, mixed upside down for 30 s, and allowed to stand for 3 min. Subsequently, it was centrifuged at a temperature of 4 ° C and a rotational speed of 12000 rpm for 15 min. The solution lysate was divided into three layers, wherein the upper layer was RNA dissolved in the aqueous phase. The upper layer solution was taken and an equal volume of isopropanol was added thereto, mixed and allowed to stand at 4 ° C for 10 min. Thereafter, it was again centrifuged at a temperature of 4 ° C and a rotational speed of 12000 rpm for 10 min and the supernatant was removed. Subsequently, 1 mL of 75% ethanol was added to the precipitate and the mixture was resuspended by mixing upside down. It was then centrifuged for 10 min at a temperature of 4 ° C and a speed of 12000 rpm and the supernatant was removed, leaving a precipitate. The precipitate was then dried at room temperature for 15 min until there was no liquid on the tube wall. Subsequently, 25 μL of DEPC H ₂ O was added to dissolve the RNA to obtain an RNA solution.

(S2) Removal of DNA from cell samples

Prepare the experimental system shown below:

The above experimental system was then incubated at room temperature for 30 min, after which 300 μL of Buffer RP was added and vortexed for 15 s, followed by standing for 10 min to inactivate DNase I. Thereafter, 250 μL of absolute ethanol was added and vortexed for 15 s, followed by brief centrifugation to remove droplets on the tube wall. The RNA was then purified using a Hipure RNA column and eluted with 25 μL of DEPC H ₂ O to obtain a DNA sample after DNA removal.

(S3) Detect and assess the total RNA quality in the sample to determine the RNA quality meets the requirements

The degree of RNA degradation in the RNA sample after DNA removal was measured by 1% agarose gel electrophoresis and it was determined whether there was contamination. The results showed that the sample was non-polluting and the degree of RNA degradation was small. The purity of the RNA was then detected by an ultraviolet spectrophotometer with an OD260/280 ratio of 1.95 and a high purity. RNA integrity was then analyzed by the Agilent 2100 Bioanalyzer, showing good RNA integrity with a RIN value of 9.2.

(S4) Removal of rRNA

195 masses of 50 bp DNA probes complementary to the rRNA sequences in the cells were designed to form a DNA probe library, and then the experimental system shown below was prepared:

DNA probe library 5μg

RNA sample after DNA removal 5μg

5×hybridization buffer 5 μL (the composition is 1 M NaCl, 0.5 M Tris-HCl, pH 7.4);

Make up DEPC water to 25μL.

The above experimental system was mixed at 95 ° C for two minutes, and then the temperature of the experimental system was lowered to 45 ° C at a rate of 0.1 ° C / s to form a DNA-RNA hybrid. Subsequently, 10 μL of RNase H (5 U/μL) and 5 μL of 10×RNase H Reaction Buffer (component: 500 mM Tris-HCl, 750 mM KCL, 30 mM MgCl ₂ , 100 mM DTT, pH=8.3) which had been preheated to 37 ° C and 5 μL of DEPC water was added to the DNA-RNA hybrid. The RNA hybridized with the DNA was then digested for 30 min at 37 ° C and purified using 2.2× RNA Clean XPbeads. Then, 5 μL of Turbo DNaseI (2 U/μL) was added to the reaction system and the DNA probe was digested at 37 ° C for 30 min. Subsequently, purification was carried out using 2.2× RNA Clean XP beads to obtain rRNA-depleted RNA, and the rRNA depleted RNA concentration was measured by a Qubit fluorometer, and the concentration was found to be 21.78 ng/μL.

(S5) Removal of linear RNA

Prepare the experimental system shown below:

The above system was digested in a 37 ° C water bath for 30 min to obtain linear RNA-removed RNA, and the concentration was measured by a Qubit fluorometer, and the concentration was found to be 4.60 ng/μL.

(S6) Construction of a circular RNA library for high-throughput sequencing

(S61) RNA Fragmentation

Prepare the experimental system shown below:

5×First Strand Buffer 8μL

Linear RNA-removed RNA 10 μL

Nuclease free H ₂ O 2μL;

The above experimental system was incubated in a PCR apparatus which was programmed to stand at 94 ° C for 5 min and then immediately placed on ice to obtain fragmented RNA.

(S62) First strand synthesis:

Prepare the experimental system shown below:

The above experimental system was incubated in a PCR apparatus. The procedure of the PCR instrument was: 65 ° C for 3 min, then placed on ice, and 4 μL of Nuclease free H ₂ O, 1 μL of 100 mM DTT, 0.1 μL of 25 mM dNTPs, and 0.5 μL of SupeRase were added. -In, 0.5 μL M-MulVReverse Transcriptase and 4 μg Actinomycin D. The reaction system was then incubated in a PCR machine with the following procedures: 25 ° C for 10 min; 42 ° C for 50 min; 70 ° C for 15 min; 4 ° CHold. After purification, 38 μL of RNA Clean XP and 19 μL of 100% ethanol were added and eluted with 16 μL of Nuclease Free H ₂ O to obtain an RNA/cDNA hybrid, which was simultaneously transferred to a new tube.

(S63) Second strand synthesis

The experimental system shown below was prepared at 0 °C:

The above experimental system was incubated at 16 ° C for 2.5 h. Subsequently, 38 μL of RNA Clean XP and 19 μL of ethanol were added for purification and eluted with 32 μL of Qiagen EB to obtain dsDNA.

(S64) end repair

The experimental system shown below was prepared at 0 °C:

The above experimental system was placed in a PCR machine and kept at 20 ° C for 30 min. Subsequently, 28 μL of RNA Clean XP and 14 μL of ethanol were added and eluted with 17 μL of Nuclease Free H ₂ O to obtain dsDNA after terminal repair.

(S65) plus A tail

Prepare the experimental system shown below:

It was then incubated at 37 ° C for 30 min. Subsequently, 28 μL of AMPure XP beads and 14 μL of ethanol were added for purification and eluted with 10 μL of Nuclease Free H ₂ O to obtain A-tailed dsDNA.

(S66) Y-type Adapter connection:

The experimental system shown below was prepared at 0 °C:

The above experimental system was placed in a PCR machine and incubated at 20 ° C for 20 min, then mixed with 24 μL of "12P XP" beads and incubated at room temperature for 6 min, after which the supernatant was retained and compared with 12 μL of AMPure XP beads and 5 μL of 40 wt%. The PEG8000 was mixed and incubated for 6 min at room temperature, followed by elution with 10 μL of Nuclease Free H ₂ O to obtain an eluate, after which the eluate was mixed with 12 μL of AMPure XP, incubated again for 6 min at room temperature, and then 30 μL of Qiagen was used. The EB was eluted once to obtain a product.

(S67) Second strand digestion and library amplification

15 μL of the above product was mixed with 1 μL of Uracil DNA Glycosylase and incubated at 37 ° C for 30 min to obtain UNG digested DNA. The experimental system shown below was then prepared at 0 °C:

The above experimental system was subjected to near-PCR amplification, and the cycle conditions were: 94 ° C for 30 s; (98 ° C for 10 s; 65 ° C for 30 s; 72 ° C for 30 s) for 15 cycles; 72 ° C for 5 min; and then the temperature of the experimental system was maintained at 4 ° C. Subsequently, 43 μL of AMPure XP beads was added for purification and eluted with 12 μL of Qiagen EB to obtain a library.

(S68) Quality Inspection and Preparation Indexing Library

The library concentration was first measured using a Qubit fluorometer and found to be 4.06 ng/μL. The library quality was then measured using an Agilent 2100 Bioanalyzer. The results are shown in Figure 2. The results showed that the insert size was acceptable, the peak shape was single, and the peak value was around 200-500 bp. Different indexed libraries are then mixed according to the detected concentration. Start using the Qubit fluorometer to detect the concentration of the library. Sequencing on the machine.

Example 2 Construction of High-throughput Sequencing Library of Circular RNA in Fresh Liver Cancer Tissue Samples

(S1) Extraction of total RNA from fresh liver cancer tissue samples

50 g of fresh liver cancer tissue sample was taken, washed once with PBS at 4 ° C, and the tissue was cut. Then 1 mL Trizol was added and homogenized 15 times with a hand-held high speed disperser. The sample was then collected into an EP tube and 200 μL of chloroform was added, mixed upside down for 30 s and allowed to stand for 3 min. Subsequently, it was centrifuged at a temperature of 4 ° C and a rotational speed of 12000 rpm for 15 min. The solution lysate was divided into three layers, wherein the upper layer was RNA dissolved in the aqueous phase. The upper layer solution was taken and an equal volume of isopropanol was added thereto, mixed and allowed to stand for 10 min. Thereafter, it was again centrifuged at a temperature of 4 ° C and a rotational speed of 12000 rpm for 10 min and the supernatant was removed. Subsequently, 1 mL of 75% ethanol was added to the precipitate and the mixture was resuspended by mixing upside down. It was then centrifuged for 10 min at a temperature of 4 ° C and a speed of 12000 rpm and the supernatant was removed, leaving a precipitate. The precipitate was then dried at room temperature for 15 min until there was no liquid on the tube wall. Subsequently, 30 μL of DEPC H ₂ O was added to dissolve the RNA to obtain an RNA solution.

(S2) Removal of DNA from fresh tissue samples

Prepare the experimental system shown below:

The above experimental system was then incubated at room temperature for 30 min, after which 300 μL of Buffer RP was added and vortexed for 15 s, followed by standing for 10 min to inactivate DNase I. Thereafter, 250 μL of absolute ethanol was added and vortexed for 15 s, followed by centrifugation to remove droplets on the tube wall. The RNA was then purified using a Hipure RNA column and eluted with 25 μL of DEPC H ₂ O to obtain a DNA sample after DNA removal.

(S3) Detect and assess the total RNA quality in the sample to determine the RNA quality meets the requirements

The degree of RNA degradation in the RNA samples after DNA removal was measured by 1% agarose gel electrophoresis and the contamination was determined. The results showed that the samples were non-contaminating and the degree of RNA degradation was small. The purity of the RNA was subsequently detected by an ultraviolet spectrophotometer with an OD260/280 ratio of 2.05 and a high purity. RNA integrity was then analyzed by the Agilent 2100 Bioanalyzer, showing good RNA integrity with RIN of 7.1.

(S4) Removal of rRNA

Designed to mix 195 masses of 50 bp DNA probes complementary to the rRNA sequence in fresh tissue to form The DNA probe library is then prepared with the experimental system shown below:

DNA probe library 5μg

RNA sample after DNA removal 5μg

5×hybridization buffer 5 μL (the composition is 1 M NaCl, 0.5 M Tris-HCl, pH 7.4)

Make up DEPC water to 25μL.

The above experimental system was mixed at 95 ° C for two minutes, and then the temperature of the experimental system was lowered to 45 ° C at a rate of 0.1 ° C / s to form a DNA-RNA hybrid. Subsequently, 10 μL of RNase H (5 U/μL) and 5 μL of 10×RNase H Reaction Buffer (component: 500 mM Tris-HCl, 750 mM KCL, 30 mM MgCl ₂ , 100 mM DTT, pH=8.3) which had been preheated to 37 ° C and 5 μL of DEPC water was added to the DNA-RNA hybrid. The RNA hybridized with the DNA was then digested for 30 min at 37 ° C and purified using 2.2× RNA Clean XP beads. Then, 5 μL of Turbo DNaseI (2 U/μL) was added to the reaction system and the DNA probe was digested at 37 ° C for 30 min. Subsequently, purification was carried out using 2.2×RNA Clean XP beads to obtain rRNA-depleted RNA, and the rRNA depleted RNA concentration was measured by a Qubit fluorometer, and the concentration was 24.8 ng/μL.

(S5) Removal of linear RNA

Prepare the experimental system shown below:

The above system was digested in a 37 ° C water bath for 30 min to obtain RNA for linear RNA removal, and the concentration was measured by a Qubit fluorometer, and the concentration was 8.2 ng/μL.

(S6) Construction of a circular RNA library for high-throughput sequencing

(S61) RNA Fragmentation

Prepare the experimental system shown below:

5×First Strand Buffer 8μL

Linear RNA-removed RNA 10 μL

Nuclease free H ₂ O 2μL;

The above experimental system was incubated in a PCR apparatus which was programmed to stand at 94 ° C for 5 min and then immediately placed on ice to obtain fragmented RNA.

(S62) First strand synthesis:

Prepare the experimental system shown below:

The above experimental system was incubated in a PCR apparatus. The procedure of the PCR instrument was: 65 ° C for 3 min, then placed on ice, and 4 μL of Nuclease free H ₂ O, 1 μL of 100 mM DTT, 0.1 μL of 25 mM dNTPs, and 0.5 μL of SupeRase were added. -In, 0.5 μL M-MulVReverse Transcriptase and 4 μg Actinomycin D. The reaction system was then incubated in a PCR machine with a procedure of: 25 ° C for 10 min; -42 ° C for 50 min; -70 ° C for 15 min; - 4 ° CHold. After purification, 38 μL of RNA Clean XP and 19 μL of 100% ethanol were added and eluted with 16 μL of Nuclease Free H ₂ O to obtain an RNA/cDNA hybrid, which was simultaneously transferred to a new tube.

(S63) Second strand synthesis

The experimental system shown below was prepared at 0 °C:

The above experimental system was incubated at 16 ° C for 2.5 h. Subsequently, 38 μL of RNA Clean XP and 19 μL of ethanol were added for purification and eluted with 32 μL of Qiagen EB to obtain dsDNA.

(S64) end repair

The experimental system shown below was prepared at 0 °C:

The above experimental system was placed in a PCR machine and kept at 20 ° C for 30 min. Subsequently, 28 μL of RNA Clean XP and 14 μL of ethanol were added and eluted with 17 μL of Nuclease Free H ₂ O to obtain dsDNA after terminal repair.

(S65) plus A tail

Prepare the experimental system shown below:

It was then incubated at 37 ° C for 30 min. Subsequently, 28 μL of AMPure XP beads and 14 μL of ethanol were added for purification and eluted with 10 μL of Nuclease Free H ₂ O to obtain A-tailed dsDNA.

(S66) Y-type Adapter connection:

The experimental system shown below was prepared at 0 °C:

The above experimental system was placed in a PCR machine and incubated at 20 ° C for 20 min, then mixed with 24 μL of "12P XP" beads and incubated at room temperature for 6 min, after which the supernatant was retained and compared with 12 μL of AMPure XP beads and 5 μL of 40 wt%. The PEG8000 was mixed and incubated for 6 min at room temperature, followed by elution with 10 μL of Nuclease Free H ₂ O to obtain an eluate, after which the eluate was mixed with 12 μL of AMPure XP, incubated again for 6 min at room temperature, and then 30 μL of Qiagen was used. The EB was eluted once to obtain a product.

(S67) Second strand digestion and library amplification

15 μL of the above product was mixed with 1 μL of Uracil DNA Glycosylase and incubated at 37 ° C for 30 min to obtain UNG digested DNA. The experimental system shown below was then prepared at 0 °C:

The above experimental system was amplified by near PCR, and the cycle conditions were: 94 ° C for 30 s; (-98 ° C for 10 s; -65 ° C for 30 s; -72 ° C 30 s) cycle 15 cycles; -72 ° C for 5 min; then maintain the temperature of the experimental system at -4 °C. Subsequently, 43 μL of AMPure XP beads was added for purification and eluted with 12 μL of Qiagen EB to obtain a library.

(S68) Quality Inspection and Preparation Indexing Library

The library concentration was first measured using a Qubit fluorometer and found to be 5.16. Then the quality of the library was detected using an Agilent 2100 Bioanalyzer. The results are shown in Figure 2. The size of the insert is acceptable, the peak shape is single, and the peak value is about 200-500 bp. Different indexed libraries are then mixed according to the detected concentration. The sequencing of the library was started with a Qubit fluorometer and the sequencing was started.

Example 3 Construction of a High-throughput Sequencing Library of Circular RNA in FFPE Samples

(S1) Extraction of total RNA from FFPE samples

The FFPE samples were sectioned into 8 μm thick pieces, and 7 sections were immediately transferred to a 1.5 mL centrifuge tube. Then 1 mL of xylene was added and vortexed vigorously for 30 s and centrifuged at 14,000 rpm for 2 min. Then, 1 mL of ethanol was added to the sample and vortexed for 30 s, and then centrifuged at 14,000 rpm for 2 min, the supernatant was removed, and the precipitate was retained. It was dried at 37 ° C for 15 min to remove ethanol. Then, 200 μL of the lysate and 20 μl of Proteinase K were added to the pellet and vortexed to mix. Thereafter, the mixture was subjected to a water bath at a temperature of 55 ° C for 15 min, followed by a water bath at a temperature of 80 ° C for 15 min. After a brief low-speed centrifugation, add 200 μL of buffer to the sample and vortex for 20 s. 600 μL of absolute ethanol was then added to the sample and vortexed for 20 s. Thereafter, the mixed solution was transferred to an adsorption column, and centrifuged at 8000 rpm for 50 s, the effluent was discarded, and the column was placed in a collection tube. Then, 500 μL of the rinse liquid was added to the column, and then centrifuged at 8000 rpm for 50 s, the effluent was discarded, and the column was placed in a collection tube. Thereafter, 500 μL of the rinse liquid was added to the column, followed by centrifugation at 8000 rpm for 50 s. Discard the effluent and install the column in the collection tube. It was then centrifuged at 13,000 rpm for 3 min to dry the column matrix and transfer the column to a new 1.5 mL centrifuge tube. Then, 30 μL of DEPC water was added to the center of the column of the column and allowed to stand for 2 min, and then centrifuged at 13,000 rpm for 1 min to obtain an RNA solution.

(S2) Removal of DNA from FFPE samples

Prepare the experimental system shown below:

The above experimental system was then incubated at room temperature for 30 min, after which 300 μL of Buffer RP was added and vortexed for 15 s, followed by standing for 10 min to inactivate DNase I. Thereafter, 250 μL of absolute ethanol was added and vortexed for 15 s, followed by centrifugation to remove droplets on the tube wall. The RNA was then purified using a Hipure RNA column and eluted with 25 μL of DEPC H ₂ O to obtain a DNA sample after DNA removal.

(S3) Detect and assess the total RNA quality in the sample to determine the RNA quality meets the requirements

The degree of RNA degradation in the RNA samples after DNA removal was measured by 1% agarose gel electrophoresis and the presence of contamination was determined. The results showed that the sample RNA was significantly degraded. The purity of the RNA was then determined using an ultraviolet spectrophotometer with an OD260/280 ratio of 2.37. RNA integrity was then analyzed by the Agilent 2100 Bioanalyzer, showing poor RNA integrity with a RIN of 4.5.

(S4) Removal of rRNA

195 masses of 50 bp DNA probes complementary to the rRNA sequence in the FFPE sample were designed to form a DNA probe library, and then the experimental system shown below was prepared:

DNA probe library 5μg

RNA sample after DNA removal 5μg

5×hybridization buffer 5 μL (composition of 1 M NaCl, 0.5 M Tris-HCl, pH 7.4)

Make up DEPC water to 25μL.

The above experimental system was mixed at 95 ° C for two minutes, and then the temperature of the experimental system was lowered to 45 ° C at a rate of 0.1 ° C / s to form a DNA-RNA hybrid. Subsequently, 10 μL of RNase H (5 U/μL) and 5 μL of 10×RNase H Reaction Buffer (component: 500 mM Tris-HCl, 750 mM KCL, 30 mM MgCl ₂ , 100 mM DTT, pH=8.3) which had been preheated to 37 ° C and 5 μL of DEPC water was added to the DNA-RNA hybrid. The RNA hybridized with the DNA was then digested for 30 min at 37 ° C and purified using 2.2× RNA Clean XP beads. Then, 5 μL of Turbo DNaseI (2 U/μL) was added to the reaction system and the DNA probe was digested at 37 ° C for 30 min. Subsequently, purification was carried out using 2.2×RNA Clean XP beads to obtain rRNA-depleted RNA, and the rRNA depleted RNA concentration was measured by a Qubit fluorometer, and the concentration was 19.9 ng/μL.

(S5) Removal of linear RNA

Prepare the experimental system shown below:

The above system was placed in a 37 ° C water bath for 30 min to obtain linear RNA-removed RNA, while using Qubit The concentration was measured by a fluorometer, and the result showed a concentration of 3.25 ng/μL.

(S6) Construction of a circular RNA library for high-throughput sequencing

(S61) RNA Fragmentation

Prepare the experimental system shown below:

5×First Strand Buffer 8μL

Linear RNA-removed RNA 10 μL

Nuclease free H ₂ O 2μL;

The above experimental system was incubated in a PCR apparatus which was programmed to stand at 94 ° C for 5 min and then immediately placed on ice to obtain fragmented RNA.

(S62) First strand synthesis:

Prepare the experimental system shown below:

The above experimental system was incubated in a PCR apparatus. The procedure of the PCR instrument was: 65 ° C for 3 min, then placed on ice, and 4 μL of Nuclease free H ₂ O, 1 μL of 100 mM DTT, 0.1 μL of 25 mM dNTPs, and 0.5 μL of SupeRase were added. -In, 0.5 μL M-MulVReverse Transcriptase and 4 μg Actinomycin D. The reaction system was then incubated in a PCR machine with a procedure of: 25 ° C for 10 min; -42 ° C for 50 min; -70 ° C for 15 min; - 4 ° CHold. Thereafter, 38 μL of RNA Clean XP and 19 μL of 100% ethanol were added for purification, and eluted with 16 μL of Nuclease Free H ₂ O to obtain an RNA/cDNA hybrid, which was simultaneously transferred to a new tube.

(S63) Second strand synthesis

The experimental system shown below was prepared at 0 °C:

The above experimental system was incubated at 16 ° C for 2.5 h. Subsequently, 38 μL of RNA Clean XP and 19 μL of ethanol were added for purification and eluted with 32 μL of Qiagen EB to obtain dsDNA.

(S64) end repair

The experimental system shown below was prepared at 0 °C:

The above experimental system was placed in a PCR machine and kept at 20 ° C for 30 min. Subsequently, 28 μL of RNA Clean XP and 14 μL of ethanol were added and eluted with 17 μL of Nuclease Free H ₂ O to obtain dsDNA after terminal repair.

(S65) plus A tail

Prepare the experimental system shown below:

It was then incubated at 37 ° C for 30 min. Subsequently, 28 μL of AMPure XP beads and 14 μL of ethanol were added for purification and eluted with 10 μL of Nuclease Free H ₂ O to obtain A-tailed dsDNA.

(S66) Y-type Adapter connection:

The experimental system shown below was prepared at 0 °C:

The above experimental system was placed in a PCR machine and incubated at 20 ° C for 20 min, then mixed with 24 μL of "12P XP" beads and incubated at room temperature for 6 min, after which the supernatant was retained and compared with 12 μL of AMPure XP beads and 5 μL of 40 wt%. The PEG8000 was mixed and incubated for 6 min at room temperature, followed by elution with 10 μL of Nuclease Free H ₂ O to obtain an eluate, after which the eluate was mixed with 12 μL of AMPure XP, incubated again for 6 min at room temperature, and then 30 μL of Qiagen was used. The EB was eluted once to obtain a product.

(S67) Second strand digestion and library amplification

15 μL of the above product was mixed with 1 μL of Uracil DNA Glycosylase and incubated at 37 ° C for 30 min to obtain UNG digested DNA. The experimental system shown below was then prepared at 0 °C:

The above experimental system was subjected to near-PCR amplification, and the cycle conditions were: 94 ° C for 30 s; (98 ° C for 10 s; 65 ° C for 30 s; 72 ° C for 30 s) for 15 cycles; 72 ° C for 5 min; and then the temperature of the experimental system was maintained at 4 ° C. Subsequently, 43 μL of AMPure XP beads was added for purification and eluted with 12 μL of Qiagen EB to obtain a library.

(S68) Quality Inspection and Preparation Indexing Library

The library concentration was first measured using a Qubit fluorometer and found to be 5.24 ng/μL. Then the quality of the library was detected using an Agilent 2100 Bioanalyzer. The results are shown in Figure 2. The size of the insert is acceptable, the peak shape is single, and the peak value is about 200-500 bp. Different indexed libraries are then mixed according to the detected concentration. After the library concentration was detected by the Qubit fluorometer, the sequencing of the machine was started.

Sequencing Example Bioinformatics Analysis Sequencing Data

The circular RNA high-throughput sequencing library constructed in Examples 1-3 and the literature reported method (see Molecular Cell 56, 55-66, October 2, 2014) were constructed on a Illumina sequencing platform. Sequencing. The results are shown in Table 1.

Table 1 Bioinformatics analysis of the method of the present invention and the sequencing method of the literature report method

The results of the bioinformatics analysis of the sequencing data are shown in Table 1. In contrast, the method provided in the examples of the present invention can further remove the rRNA in the sample, and the rRNA residual ratio in the data is <0.1%, and the rRNA residue in the literature report method. At 5%, the RNA sample with severe degradation such as FFPE is as high as 37%, and the amount of data is seriously wasted. The method of the present invention significantly increases the amount of valid sequencing data of the circular RNA. In terms of the amount of circular RNA detection, samples with good RNA integrity (cell samples) have more cyclic RNA detected by literature reports, but samples with poor RNA quality, especially FFPE samples, are significantly superior to those reported in the literature. method. In addition, the method of the present invention is used to construct a high-throughput sequencing library of circular RNAs, which has a standard operating procedure, optimized conditions, stable results, and the cost is only 1/3 of the reported method. From the sequencing results, 20 circular RNAs were randomly selected for QPCR verification, and the positive rate of the verification results was 100% (Fig. 3), which strongly confirmed the reliability of the method of the present invention.

It can be confirmed that the method used in the examples of the present invention can significantly enrich the circular RNA in the sample, greatly reduce the amount of cyclic RNA sequencing data, reduce the cost, and reduce the waste of human and material resources.

Variations and modifications of the above-described embodiments may also be made by those skilled in the art in light of the above disclosure. Therefore, the present invention is not limited to the specific embodiments disclosed and described, and the modifications and variations of the invention are intended to fall within the scope of the appended claims. In addition, although specific terms are used in the specification, these terms are merely for convenience of description and do not limit the invention.

Claims (9)

A method for constructing a circular RNA high-throughput sequencing library, which in turn comprises the following steps: (S1) extracting total RNA in the sample; (S2) removing DNA from the sample; (S3) Detecting and assessing the total RNA quality in the sample to determine that the RNA quality meets the requirements; (S4) removing rRNA; (S5) removing linear RNA; (S6) Construction of a circular RNA library for high throughput sequencing. The method according to claim 1, wherein the detecting the total RNA quality in the sample in the step (S3) comprises the following steps: (S31) Using 1% agarose gel electrophoresis to detect the degree of RNA degradation and whether there is contamination; (S32) detecting the purity of the RNA by using an ultraviolet spectrophotometer, that is, an OD260/280 ratio; (S33) Detection of RNA integrity. The construction method according to claim 1, wherein the step (S4) of removing the rRNA comprises the following steps: (S41) mixing a mass of a DNA probe complementary to the rRNA sequence to form a DNA probe library; (S42) mixing and obtaining hybridization of the obtained DNA probe library with RNA which has been tested and evaluated for total RNA quality in the sample to determine the quality of the RNA, to form a DNA-RNA hybrid; (S43) digesting the obtained DNA-RNA hybrid with RNase H; (S44) digesting the DNA probe with DNase I to obtain rRNA depleted RNA; (S45) The concentration of rRNA depleted RNA was measured by a fluorometer. The construction method according to claim 3, wherein the weight ratio of the DNA probe library to RNA used in the step (S42) is (1-2): 1, preferably 1:1. The method according to claim 3, wherein the hybrid hybridization in the step (S42) is carried out under conditions of hybridization at a temperature of 95 ° C for 2 minutes and then slowly at a temperature of 0.1 ° C / s. Reduced to 45 ° C. The construction method according to claim 3, wherein the DNA-RNA hybrid obtained by digesting with RNase H in the step (S43) is subjected to digestion at a temperature of 37 ° C for 30 minutes. The construction method according to claim 1, wherein the constructing the circular RNA library for high-throughput sequencing in the step (S6) comprises the following steps in sequence: (S61) RNA fragmentation; (S62) First strand synthesis: using reverse transcriptase plus random primers; (S63) second strand synthesis: digesting the RNA strand in the RNA/cDNA hybrid using RNase H, and then synthesizing the second strand by DNA polymerase I; (S64) End repair: formation of blunt-end dsDNA using End Repair Mix and addition of a phosphate group at the 5' end; (S65) plus A tail: use Klenow 3'-5'exo- to add A tail at the end of dsDNA; (S66) Y-type Adapter connection: use T4 DNA Ligase to connect Adapter and d-DNA with A tail; (S67) second strand digestion and library amplification: digestion of the second strand with UNG enzyme; (S68) Quality inspection and preparation of the index library. The construction method according to claim 7, wherein the conditions for RNA fragmentation in the step (S61) are fragmentation at a temperature of 90 to 96 ° C, preferably 94 ° C, for 3 to 6 minutes, preferably 5 minutes. A kit for constructing a circular RNA high-throughput sequencing library, characterized in that it comprises: (1) a circular RNA enrichment reagent, DNase I, RNase H, RNaseR; (2) species-specific DNA probes; (3) various enzymes required for library construction: DNA polymerase I, Large Fragment, T4 Polynucleotide Kinase, Klenow Fragment, Escherichia coli UDG, T4 DNA Ligase, Phusion High-Fidelity DNA Polymerase; (4) magnetic beads, dNTP, Nuclease Free Water, dUTP, linkers, primers, Wherein the species-specific DNA probe is complementary to the rRNA sequence of the corresponding species.

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