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WO2018149091A1 - 一种环状rna高通量测序文库的构建方法及其试剂盒 - Google Patents

一种环状rna高通量测序文库的构建方法及其试剂盒 Download PDF

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WO2018149091A1
WO2018149091A1 PCT/CN2017/093865 CN2017093865W WO2018149091A1 WO 2018149091 A1 WO2018149091 A1 WO 2018149091A1 CN 2017093865 W CN2017093865 W CN 2017093865W WO 2018149091 A1 WO2018149091 A1 WO 2018149091A1
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rna
library
dna
throughput sequencing
sample
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French (fr)
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赖炳权
罗景燕
何重华
何铭辉
李伟琴
唐毅
黄鸿昌
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Guangzhou Forevergen Biotechnology Co Ltd
Guangzhou Forevergen Health Technology Co Ltd
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Guangzhou Forevergen Biotechnology Co Ltd
Guangzhou Forevergen Health Technology Co Ltd
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    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • 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.
  • a circular RNA 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.
  • circular RNA is associated with diseases such as neurodevelopment, atherosclerosis, myotonic dystrophy, cancer, etc.
  • diseases such as neurodevelopment, atherosclerosis, myotonic dystrophy, cancer, etc.
  • 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.
  • 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
  • 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.
  • the present invention provides a method for constructing a circular RNA high-throughput sequencing library, which in turn comprises the following steps:
  • 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.
  • the removal of DNA in the sample in the step (S2) is carried out by DNase I digestion.
  • detecting the total RNA quality in the sample in the step (S3) comprises the following steps:
  • the detection of RNA integrity in the step (S33) is performed using an Agilent 2100 bioanalyzer.
  • OD optical density
  • 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.
  • the removal of the rRNA in the step (S4) is performed by the RNase H digestion method.
  • the step (S4) of removing the rRNA sequentially comprises the following steps:
  • the weight ratio of the DNA probe library to the RNA used in the step (S42) is (1-2): 1, preferably 1:1. .
  • 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.
  • 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.
  • the fluorometer used to determine the concentration of rRNA-depleted RNA in the step (S45) is performed by a Qubit fluorometer.
  • 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.
  • 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.
  • the construction of the circular RNA library for high-throughput sequencing in the step (S6) is carried out by the dUTP method.
  • constructing the circular RNA library for high-throughput sequencing in the step (S6) sequentially comprises the following steps:
  • 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.
  • the peak after RNA fragmentation in the step (S61) is located at 300-350 bp.
  • 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.
  • 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.
  • 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.
  • the step (S68) of the quality inspection and preparation of the indexing library sequentially comprises the following steps:
  • 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.
  • kits for constructing a circular RNA high-throughput sequencing library comprising:
  • RNA enrichment reagent DNase I, RNase H, RNaseR;
  • 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.
  • the present invention has the following advantages and benefits:
  • 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.
  • FIG. 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
  • Example 3 is a result of QPCR verification after selecting 20 circular RNAs after sequencing of the cell sample of Example 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:
  • the total RNA extracted from the sample is performed using the Trizol method, which is particularly suitable for tissue and cell samples.
  • removal of DNA from the sample is carried out using the DNase I digestion method.
  • detecting and assessing the total RNA quality in the sample comprises the following steps:
  • the integrity of the RNA detected in the step (S33) was performed using an Agilent 2100 Bioanalyzer.
  • Removal of rRNA was performed using the RNase H digestion method.
  • 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.
  • step (S45) 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.
  • RNA removal 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.
  • 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:
  • 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);
  • 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.
  • the Adapter end of the Y-type Adapter is connected with T by a phosphorothioate bond to prevent formation of an Adapter dimer.
  • 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:
  • 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 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 ⁇ L of DEPC H 2 O was added to dissolve the RNA to obtain an RNA solution.
  • RNA integrity was then analyzed by the Agilent 2100 Bioanalyzer, showing good RNA integrity with a RIN value of 9.2.
  • RNase H 5 U/ ⁇ L
  • RNA hybridized with the DNA was then digested for 30 min at 37 ° C and purified using 2.2 ⁇ RNA Clean XPbeads. Then, 5 ⁇ L of Turbo DNaseI (2 U/ ⁇ L) 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/ ⁇ L.
  • RNA-removed RNA 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/ ⁇ L.
  • 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.
  • 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 ⁇ L of Nuclease free H 2 O, 1 ⁇ L of 100 mM DTT, 0.1 ⁇ L of 25 mM dNTPs, and 0.5 ⁇ L of SupeRase were added.
  • -In 0.5 ⁇ L M-MulVReverse Transcriptase and 4 ⁇ g 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.
  • RNA Clean XP 38 ⁇ L of RNA Clean XP and 19 ⁇ L of 100% ethanol were added and eluted with 16 ⁇ L of Nuclease Free H 2 O to obtain an RNA/cDNA hybrid, which was simultaneously transferred to a new tube.
  • RNA Clean XP 38 ⁇ L of RNA Clean XP and 19 ⁇ L of ethanol were added for purification and eluted with 32 ⁇ L of Qiagen EB to obtain dsDNA.
  • the above experimental system was placed in a PCR machine and incubated at 20 ° C for 20 min, then mixed with 24 ⁇ L of "12P XP" beads and incubated at room temperature for 6 min, after which the supernatant was retained and compared with 12 ⁇ L of AMPure XP beads and 5 ⁇ L of 40 wt%.
  • the PEG8000 was mixed and incubated for 6 min at room temperature, followed by elution with 10 ⁇ L of Nuclease Free H 2 O to obtain an eluate, after which the eluate was mixed with 12 ⁇ L of AMPure XP, incubated again for 6 min at room temperature, and then 30 ⁇ L of Qiagen was used.
  • the EB was eluted once to obtain a product.
  • 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 ⁇ L of AMPure XP beads was added for purification and eluted with 12 ⁇ L of Qiagen EB to obtain a library.
  • the library concentration was first measured using a Qubit fluorometer and found to be 4.06 ng/ ⁇ L.
  • 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.
  • 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 ⁇ L of DEPC H 2 O was added to dissolve the RNA to obtain an RNA solution.
  • RNA integrity was then analyzed by the Agilent 2100 Bioanalyzer, showing good RNA integrity with RIN of 7.1.
  • DNA probe library is then prepared with the experimental system shown below:
  • RNase H 5 U/ ⁇ L
  • 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 ⁇ L of Turbo DNaseI (2 U/ ⁇ L) 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/ ⁇ L.
  • RNA for linear RNA removal 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/ ⁇ L.
  • 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.
  • 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 ⁇ L of Nuclease free H 2 O, 1 ⁇ L of 100 mM DTT, 0.1 ⁇ L of 25 mM dNTPs, and 0.5 ⁇ L of SupeRase were added.
  • -In 0.5 ⁇ L M-MulVReverse Transcriptase and 4 ⁇ g 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.
  • RNA Clean XP 38 ⁇ L of RNA Clean XP and 19 ⁇ L of 100% ethanol were added and eluted with 16 ⁇ L of Nuclease Free H 2 O to obtain an RNA/cDNA hybrid, which was simultaneously transferred to a new tube.
  • RNA Clean XP 38 ⁇ L of RNA Clean XP and 19 ⁇ L of ethanol were added for purification and eluted with 32 ⁇ L of Qiagen EB to obtain dsDNA.
  • the above experimental system was placed in a PCR machine and incubated at 20 ° C for 20 min, then mixed with 24 ⁇ L of "12P XP" beads and incubated at room temperature for 6 min, after which the supernatant was retained and compared with 12 ⁇ L of AMPure XP beads and 5 ⁇ L of 40 wt%.
  • the PEG8000 was mixed and incubated for 6 min at room temperature, followed by elution with 10 ⁇ L of Nuclease Free H 2 O to obtain an eluate, after which the eluate was mixed with 12 ⁇ L of AMPure XP, incubated again for 6 min at room temperature, and then 30 ⁇ L of Qiagen was used.
  • the EB was eluted once to obtain a product.
  • 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 ⁇ L of AMPure XP beads was added for purification and eluted with 12 ⁇ L of Qiagen EB to obtain a 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.
  • the FFPE samples were sectioned into 8 ⁇ m 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 ⁇ L of the lysate and 20 ⁇ l of Proteinase K were added to the pellet and vortexed to mix.
  • 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 ⁇ L of buffer to the sample and vortex for 20 s. 600 ⁇ L 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.
  • RNA solution 500 ⁇ L 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 ⁇ L 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 ⁇ L 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.
  • RNA integrity was then analyzed by the Agilent 2100 Bioanalyzer, showing poor RNA integrity with a RIN of 4.5.
  • RNase H 5 U/ ⁇ L
  • 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 ⁇ L of Turbo DNaseI (2 U/ ⁇ L) 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/ ⁇ L.
  • 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/ ⁇ L.
  • 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.
  • 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 ⁇ L of Nuclease free H 2 O, 1 ⁇ L of 100 mM DTT, 0.1 ⁇ L of 25 mM dNTPs, and 0.5 ⁇ L of SupeRase were added.
  • -In 0.5 ⁇ L M-MulVReverse Transcriptase and 4 ⁇ g 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.
  • RNA Clean XP 38 ⁇ L of RNA Clean XP and 19 ⁇ L of 100% ethanol were added for purification, and eluted with 16 ⁇ L of Nuclease Free H 2 O to obtain an RNA/cDNA hybrid, which was simultaneously transferred to a new tube.
  • RNA Clean XP 38 ⁇ L of RNA Clean XP and 19 ⁇ L of ethanol were added for purification and eluted with 32 ⁇ L of Qiagen EB to obtain dsDNA.
  • the above experimental system was placed in a PCR machine and incubated at 20 ° C for 20 min, then mixed with 24 ⁇ L of "12P XP" beads and incubated at room temperature for 6 min, after which the supernatant was retained and compared with 12 ⁇ L of AMPure XP beads and 5 ⁇ L of 40 wt%.
  • the PEG8000 was mixed and incubated for 6 min at room temperature, followed by elution with 10 ⁇ L of Nuclease Free H 2 O to obtain an eluate, after which the eluate was mixed with 12 ⁇ L of AMPure XP, incubated again for 6 min at room temperature, and then 30 ⁇ L of Qiagen was used.
  • the EB was eluted once to obtain a product.
  • 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 ⁇ L of AMPure XP beads was added for purification and eluted with 12 ⁇ L of Qiagen EB to obtain a library.
  • the library concentration was first measured using a Qubit fluorometer and found to be 5.24 ng/ ⁇ L. 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.
  • the results of the bioinformatics analysis of the sequencing data are shown in Table 1.
  • 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.
  • 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.
  • samples with good RNA integrity cell samples
  • samples with poor RNA quality, especially FFPE samples are significantly superior to those reported in the literature. method.
  • 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.
  • 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.

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Abstract

环状RNA高通量测序文库的构建方法及其试剂盒，构建方法依次包括以下步骤：(S1)提取样品中的总RNA；(S2)去除样品中的DNA；(S3)检测并评定样品中的总RNA质量，确定RNA质量符合要求；(S4)去除rRNA；(S5)去除线性RNA；(S6)构建用于高通量测序的环状RNA文库。环状RNA高通量测序文库的构建方法高效、稳定，rRNA残留比例低，环状RNA检出效率高，数据重复性较好，测序结果验证成功率高，尤其适用于FFPE组织等RNA质量差的样品。

Description

一种环状RNA高通量测序文库的构建方法及其试剂盒 技术领域
本发明属于分子生物学领域，具体涉及一种环状RNA高通量测序文库的构建方法及其试剂盒。
背景技术
环状RNA(ciucular RNA,circRNA)是一种区别于传统线性RNA的RNA家族新成员，其为不具有5’末端帽子和3’末端poly(A)尾巴且以共价键形成环形结构的非编码RNA分子。早在1980年，环状RNA就已经被发现，但是在很长的一段时间内，由于研究技术水平的限制，环状RNA被认为是错误可变剪切而形成的副产品，属于自然界中一种极罕见的现象，且甚至被当做遗传意外或实验人为因素所致，并未引起学术界重视。随着深度RNA测序和规模化生物信息技术的发展，研究者才发现在生物体内存在大量环状RNA分子。2012年Salzman等人通过高通量测序的方法发现了环状RNA广泛存在人的转录组当中，从而引起人们对环状RNA研究的兴趣。2013年Hansen等人发现CDR1as/CiRS-7可以作为miR-7sponge调控基因表达，证实了环状RNA在人体内的重要作用。最新研究表明环状RNA具有闭合环状结构，主要通过非典型可变剪切加工产生，广泛存在于各种生物细胞中，具有结构稳定，难以被RNA酶降解、表达丰度高、物种间保守性好，表达具有组织及时空特异性等特征。目前已有研究显示环状RNA跟神经发育、动脉粥样硬化、强直性肌营养不良、癌症等疾病相关，并且在人唾液和血液中检测到环状RNA的存在，则表明环状RNA可以在血液，尿液，腹水等临床标本中稳定存在，这些特征使得环状RNA在新型疾病诊断与治疗方法的开发应用上具有广阔的前景。
CN 104388548 A公开了一种高通量环状RNA测序的方法，其包括：人工合成外源线性ERCC-0004-RNA和外源环状ERCC-0013-RNA；对外源环状ERCC-0013-RNA进行质量控制；将外源线性ERCC-0004-RNA和外源环状ERCC-0013-RNA按等摩尔比混合，得到混合外源RNA；向待测序样本的总RNA中加入混合外源RNA，得到第一混合物；去除第一混合物中的核糖体RNA，得到第二混合物；对第二混合物进行3’末端生物素标记，并去除标记上生物素的套索RNA及线性RNA，得到第三混合物；去除第三混合物中的线性RNA，得到第四混 合物；对第四混合物进行标准的高通量转录组测序并对测序数据进行生物学分析。该方法步骤复杂，且需要预先合成外源RNA。
由于环状RNA无法直接分离，故而迄今文献中对于环状RNA文库构建并没有一个标准的方法，大多数研究中采用去除rRNA并去除线性RNA，然后再构建用于高通量测序文库的方法，也有一小部分研究是去除rRNA后直接建库，或者去除线性RNA后直接建库，还有极小一部分研究是采用去除PolyA+RNA后建库的方法。这些方法存在rRNA去除不彻底，数据重复性差等缺点。因此，如何高效、稳定地构建用于环状RNA高通量测序的文库，仍然是亟待解决的一个技术难题。
此外，目前市场上还没有专门的环状RNA建库试剂盒，需要科研人员从QIAGEN、Illumina、Backman、NEB、Life technology等多家公司分别购买RNA提取、rRNA去除、线性RNA去除、文库构建等产品，然后摸索合适的条件进行建库。这种多家试剂组合建库一方面价格昂贵，另一方面需要有较高实验技能的科研人员花费较长的时间去摸索最佳实验条件，严重限制了环状RNA研究的进展，因此有必要开发一款能高效、稳定地构建用于环状RNA高通量测序文库的专用试剂盒。
发明内容
鉴于上述问题，本发明发明人对环状RNA测序进行反复研究和分析，开发出一种能够高效稳定地构建环状RNA高通量测序文库的方法，该方法rRNA残留比例低，数据重复性较好，且该方法尤其适用于FFPE组织等RNA质量差的样品。
为了实现上述目的，本发明提供一种环状RNA高通量测序文库的构建方法，其依次包括以下步骤：
(S1)提取样品中的总RNA；
(S2)去除样品中的DNA；
(S3)检测并评定样品中的总RNA质量，确定RNA质量符合要求；
(S4)去除rRNA；
(S5)去除线性RNA；
(S6)构建用于高通量测序的环状RNA文库。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S1)中提取样品中的总RNA是采用Trizol法进行的，该方法特别适用于组织和细胞样品。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S2)中去除样品中的DNA是采用DNase I消化法进行的。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S3)中检测样品中总的RNA质量依次包括以下步骤：
(S31)利用1％琼脂糖凝胶电泳检测RNA降解程度以及是否有污染；
(S32)利用紫外分光光度计检测RNA的纯度，即OD260/280比值；
(S33)检测RNA的完整性。
更优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S33)中检测RNA的完整性是利用Agilent 2100生物分析仪进行的。
在本发明中，OD(optical density)表示被检测物质吸收掉的光密度，OD260/280比值是指在波长260nm和280nm处的吸光光度比值，用于判断RNA的纯度。理论上，在纯RNA的情况下OD260/280比值为2，其中OD260代表核酸的吸光度，OD280代表蛋白质的吸光度。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S4)中去除rRNA是采用RNase H消化法进行的。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S4)去除rRNA依次包括以下步骤：
(S41)将与rRNA序列互补的DNA探针等质量混合，形成DNA探针库；
(S42)将所获得DNA探针库与经过检测并评定样品中的总RNA质量确定RNA质量符合要求的RNA混合杂交，形成DNA-RNA杂交物；
(S43)用RNase H消化所获得的DNA-RNA杂交物；
(S44)用DNase I消化DNA探针，获得rRNA耗竭的RNA；
(S45)通过荧光计测定rRNA耗竭的RNA的浓度。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S42)中所用的DNA探针库与RNA的重量比为(1-2)：1，优选1:1。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S42)中混合杂交所采用的条件为在95℃的温度下杂交2分钟，然后以0.1℃/s的速度将温度缓慢地降低至45℃。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S43)中用RNase  H消化所获得的DNA-RNA杂交物所采用的条件为在37℃的温度下消化30分钟。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S45)中测定rRNA耗竭的RNA的浓度所采用的荧光计是通过Qubit荧光计进行的。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S5)中去除线性RNA是采用RNase R消化法进行的，其中RNase R与RNA的用量比为(2.5-3.5)U:1ug，优选3:1，即每1微克RNA中加入3个活性单位(U)的RNase R。
更优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S5)中去除线性RNA所采用的RNase R消化法的消化条件为在35-45℃，优选37℃的温度下消化20-40分钟，优选30分钟。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S6)中构建用于高通量测序的环状RNA文库是采用dUTP法进行的。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S6)中构建用于高通量测序的环状RNA文库依次包括以下步骤：
(S61)RNA片段化；
(S62)第一链合成：采用逆转录酶加随机引物的方法进行；
(S63)第二链合成：利用RNase H消化RNA/cDNA杂交物中的RNA链，然后通过DNA聚合酶I合成第二条链；
(S64)末端修复：利用End Repair Mix形成平末端的dsDNA并在5'端加上磷酸基团；
(S65)加A尾：利用Klenow 3'-5'exo-在dsDNA末端加A尾；
(S66)Y型Adapter连接：利用T4DNA Ligase连接Adapter和加了A尾的dsDNA；
(S67)第二链消化和文库扩增：利用UNG酶消化第二链；
(S68)质检及准备标引文库。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S61)中RNA片段化的条件为在90-96℃，优选94℃的温度下片段化3-6分钟，优选5分钟。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S61)中RNA片段化后的峰值位于300-350bp。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S62)第一链合成利用放线菌素D抑制逆转录酶的依赖DNA的DNA聚合酶活性，以保留链的方向性信息。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S66)Y型Adapter 连接中Adapter末端采用硫代磷酸酯键连接T，以防止形成Adapter二聚体，进一步优选地，连接产物纯化采用1×beads纯化一次后，0.7×/0.2×beads进行片段大小筛选，以选择合适的大小的片段。
优选地，在所述环状RNA高通量测序文库的构建方法中，所述步骤(S68)质检及准备标引文库依次包括以下步骤：
(S681)检测文库浓度；
(S682)采用Agilent 2100检测文库质量；
(S683)根据文库浓度，将不同的标引文库混合，准备上机测序。
与常规方法相比，本发明所提供的环状RNA高通量测序文库的构建方法高效、稳定，rRNA残留比例低，环状RNA检出效率高，数据重复性较好，测序结果验证成功率高，尤其适用于FFPE组织等RNA质量差的样品。
此外，本发明还提供一种环状RNA高通量测序文库构建的试剂盒，其包括：
(1)环状RNA富集试剂、DNase I、RNase H、RNaseR；
(2)种属特异的DNA探针；
(3)文库构建所需的各种酶：DNA聚合酶I、Large(Klenow)Fragment、T4 Polynucleotide Kinase(T4PNK)、Klenow Fragment、Escherichia coli UDG、T4 DNA Ligase(Rapid)、Phusion High-Fidelity DNA Polymerase；
(4)磁珠、dNTP、Nuclease Free Water、dUTP、接头、引物等，
其中，所述种属特异的DNA探针能与相应物种的rRNA序列互补。
本发明所提供的环状RNA高通量测序文库构建试剂盒操作简单，条件优化，成本低，是环状RNA研究的一个有力工具。
与现有技术相比，本发明具有以下优点和有益效果：
(1)本发明环状RNA高通量测序文库的构建方法因为建立了种属特异的DNA探针库，采用RNaseH去除与探针结合的rRNA，并采用RNaseR进一步富集环状RNA，所以rRNA残留比例低，环状RNA检出效率高，数据重复性较好，测序结果验证成功率高，尤其适用于FFPE组织等RNA质量差的样品；
(2)本发明环状RNA高通量测序文库构建试剂盒操作简单，条件优化，成本低，是环状RNA研究的一个有力工具。
附图说明
图1为本发明环状RNA高通量测序文库的构建方法的一种实施方式的流程图；
图2-1为本发明实施例1细胞样品中的环状RNA高通量测序文库的建库完成后利用Agilent 2100对文库进行质检的结果；
图2-2为本发明实施例2新鲜肝癌组织样品中的环状RNA高通量测序文库的建库完成后利用Agilent 2100对文库进行质检的结果；
图2-3为本发明实施例3FFPE样品中的环状RNA高通量测序文库的建库完成后利用Agilent 2100对文库进行质检的结果；
图3为实施例1细胞样本测序完成后挑选20个环状RNA进行QPCR验证的结果。
具体实施方式
为了使本发明的目的、技术方案和优点更加清楚，下面结合附图对本发明做进一步地详细描述。
具体地，如图1所示，本发明实施例提供一种环状RNA高通量测序文库的构建方法，其依次包括以下步骤：
S1，提取样品中的总RNA；
提取样品中的总RNA是采用Trizol法进行的，该方法特别适用于组织和细胞样品。
S2，去除样品中的DNA。
优选地，去除样品中的DNA是采用DNase I消化法进行的。
S3，检测并评定样品中的总RNA质量，确定RNA质量符合要求。
优选地，检测并评定样品中的总RNA质量依次包括以下步骤：
(S31)利用1％琼脂糖凝胶电泳检测RNA降解程度以及是否有污染；
(S32)利用紫外分光光度计检测RNA的纯度，即OD260/280比值；
(S33)检测RNA的完整性。
所述步骤(S33)中检测RNA的完整性是利用Agilent 2100生物分析仪进行的。
S4，去除rRNA。
去除rRNA是采用RNase H消化法进行的。
RNase H消化法去除rRNA依次包括以下步骤：
(S41)将与rRNA序列互补的DNA探针等质量混合，形成DNA探针库；
(S42)将所获得DNA探针库与经过检测并评定样品中总的RNA质量确定RNA质量符合要求的RNA混合杂交，形成DNA-RNA杂交物；
(S43)用RNase H消化所获得的DNA-RNA杂交物；
(S44)用DNase I消化DNA探针，获得rRNA耗竭的RNA；
(S45)通过荧光计测定rRNA耗竭的RNA的浓度。
所述步骤(S42)中所用的DNA探针库与RNA的质量比为(1-2)：1，优选1:1。
所述步骤(S42)中混合杂交所采用的条件为在95℃的温度下杂交2分钟，然后以0.1℃/s的速度将温度缓慢地降低至45℃，45℃保温5分钟。
所述步骤(S43)中用RNase H消化所获得的DNA-RNA杂交物所采用的条件为在37℃的温度下消化30分钟。
所述步骤(S45)中测定rRNA耗竭的RNA的浓度所采用的荧光计是通过Qubit荧光计进行的。
所述步骤(S45)中得到的产物进一步用安捷伦2100仪器检测证实已看不到18S，28S rRNA存在的典型峰图。
S5，去除线性RNA。
去除线性RNA是采用RNase R消化法进行的，其中RNase R与RNA的用量比为(2.5-3.5)U:1ug，优选3:1，即每1微克RNA中加入3个活性单位(U)的RNase R。
去除线性RNA所采用的RNase R消化法的消化条件为在35-45℃，优选37℃的温度下消化20-40分钟，优选30分钟。
S6，构建用于高通量测序的环状RNA文库。
构建用于高通量测序的环状RNA文库是采用dUTP法进行的。
dUTP法构建用于高通量测序的环状RNA文库依次包括以下步骤：
(S61)RNA片段化；
(S62)第一链合成：采用逆转录酶加随机引物的方法进行，原料为dNTPs(dA,dC,dG和dT各25mM)；
(S63)第二链合成：利用RNase H消化RNA/cDNA杂交物中的RNA链，然后通过DNA聚合酶I合成第二条链，原料为dUTP mix(10mM dA,dC,dG and 20mM dU)；
(S64)末端修复：利用End Repair Mix形成平末端的dsDNA并在5'端加上磷酸基团；
(S65)加A尾：利用Klenow 3'-5'exo-在dsDNA末端加A尾；
(S66)Y型Adapter连接：利用T4DNA Ligase连接Adapter和加了A尾的dsDNA；
(S67)第二链消化和文库扩增：利用UNG酶消化第二链；
(S68)质检及准备标引文库。
所述步骤(S61)中RNA片段化的条件为在90-96℃，优选94℃的温度下片段化3-6分钟，优选5分钟。
所述步骤(S61)中RNA片段化后的峰值位于300-350bp。
所述步骤(S62)第一链合成利用放线菌素D抑制逆转录酶的依赖DNA的DNA聚合酶活性，以保留链的方向性信息。
所述步骤(S66)Y型Adapter连接中Adapter末端采用硫代磷酸酯键连接T，以防止形成Adapter二聚体，进一步优选地，连接产物纯化采用1×beads纯化一次后，0.7×/0.2×beads进行片段大小筛选，以选择合适的大小的片段。
所述步骤(S68)质检及准备标引文库包括以下步骤：
(S681)Qubit荧光计检测文库浓度；
(S682)采用Agilent 2100检测文库质量；
(S683)根据文库浓度，将不同的标引文库混合，准备上机测序。
与常规方法相比，本发明所提供的环状RNA高通量测序文库的构建方法高效、稳定，rRNA残留比例低，环状RNA检出效率高，数据重复性较好，测序结果验证成功率高，尤其适用于FFPE组织等RNA质量差的样品。
为了使本发明的目的及优点更加简洁明了，本发明将用以下具体实施例进行阐明，但本发明绝非仅限于这些实施例。以下实施例仅为本发明较优选的实施例，且仅用于阐述本发明，不能理解为对本发明的范围的限制。应当指出的是，凡在本发明的实质和原则之内所做的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。因此，本发明专利的保护范围应以所附权利要求为准。
实施例1 细胞样品中的环状RNA高通量测序文库的构建
(S1)细胞样品中的总RNA的提取
取细胞样品1×107个，在4℃下用PBS洗一次，然后向6孔板每个孔中加入1mL Trizol，并用1mL枪头反复吹打10次。将样品收集到EP管内，加入200μL氯仿，上下颠倒混匀 30s后静置3min。随后再在4℃的温度和12000rpm的转速下离心15min，溶液裂解液分三层，其中上层为溶于水相的RNA。取上层溶液并向其中加入等体积的异丙醇，混匀并4℃静置10min。之后再次在4℃的温度和12000rpm的转速下离心10min并移除上清液。随后在沉淀中加入1mL 75％乙醇并上下颠倒混匀重悬沉淀。然后还在4℃的温度和12000rpm的转速下离心10min并移除上清，保留沉淀。然后将沉淀在室温下干燥15min直至管壁无液体。随后加入25μL DEPC H2O溶解RNA，以获得RNA溶液。
(S2)去除细胞样品中的DNA
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000001
然后将上述实验体系在室温下孵育30min，之后加入300μL Buffer RP并涡旋混匀15s，随后静置10min以使DNase I失活。其后加入250μL无水乙醇并涡旋混匀15s，随后进行短暂离心以去除管壁上的液滴。然后使用Hipure RNA柱子纯化RNA，并使用25μL DEPC H2O进行洗脱，得到去除DNA后的RNA样品。
(S3)检测并评定样品中的总RNA质量，确定RNA质量符合要求
利用1％琼脂糖凝胶电泳检测去除DNA后的RNA样品中的RNA降解程度并确定是否有污染，结果显示该样品无污染且RNA降解程度小。随后利用紫外分光光度计检测RNA的纯度，其中OD260/280比值为1.95，纯度较高。之后再通过Agilent 2100生物分析仪分析RNA的完整性，显示RNA完整性很好，RIN值为9.2。
(S4)去除rRNA
设计195条与细胞中的rRNA序列互补的50bp DNA探针等质量混合在一起，形成DNA探针库，然后配制如下所示的实验体系：
DNA探针库                 5μg
去除DNA后的RNA样品        5μg
5×hybridization buffer   5μL(其成分为1M NaCl，0.5M Tris-HCl，pH 7.4)；
补DEPC水至25μL。
将上述实验体系在95℃下混合两分钟，然后以0.1℃/s的速度将实验体系的温度降低至 45℃，形成DNA-RNA杂交物。随后将10μL RNase H(5U/μL)和5μL已预热至37℃的10×RNase H Reaction Buffer(其成分为：500mM Tris-HCl，750mM KCL，30mM MgCl2，100mM DTT，pH＝8.3)和5μL DEPC水加入至DNA-RNA杂交物中。然后在37℃的温度下消化与DNA杂交的RNA 30min并使用2.2×RNA Clean XPbeads进行纯化。之后向反应体系中加入5μL Turbo DNaseⅠ(2U/μL)并在37℃的条件下消化DNA探针30min。随后使用2.2×RNA Clean XP beads进行纯化，得到rRNA耗竭的RNA，同时用Qubit荧光计测定rRNA耗竭的RNA浓度，结果显示浓度为21.78ng/μL。
(S5)去除线性RNA
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000002
将上述体系置于37℃水浴中消化30min，获得线性RNA去除的RNA，同时用Qubit荧光计测定其浓度，结果显示浓度为4.60ng/μL。
(S6)构建用于高通量测序的环状RNA文库
(S61)RNA片段化
配制如下所示的实验体系：
5×First Strand Buffer     8μL
线性RNA去除的RNA           10μL
Nuclease free H2O          2μL；
将上述实验体系放入PCR仪中孵育，所述PCR仪的程序为：94℃5min，然后立刻置于冰上，得到片段化后的RNA。
(S62)第一链合成：
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000003
将上述实验体系放入PCR仪中孵育，所述PCR仪的程序为：65℃3min，然后置于冰上，并加入4μL Nuclease free H2O、1μL 100mM DTT、0.1μL 25mM dNTPs、0.5μL SupeRase-In、0.5μL M-MulVReverse Transcriptase和4μg Actinomycin D。随后将反应体系放入PCR仪中孵育，所述PCR仪的程序为：25℃10min；42℃50min；70℃15min；4℃Hold。之后加入38μL RNA Clean XP和19μL 100％ethanol进行纯化，并使用16μL Nuclease Free H2O进行洗脱，获得RNA/cDNA杂交物，同时转移至新管中。
(S63)第二链合成
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000004
将上述实验体系在16℃下孵育2.5h。随后加入38μL RNA Clean XP和19μL乙醇进行纯化并用32μL Qiagen EB进行洗脱，得到dsDNA。
(S64)末端修复
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000005
将上述实验体系放入PCR仪中并在20℃下保持30min。随后加入28μL RNA Clean XP和14μL乙醇进行并用17μL Nuclease Free H2O进行洗脱，得到末端修复后的dsDNA。
(S65)加A尾
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000006
Figure PCTCN2017093865-appb-000007
然后在37℃下孵育30min。随后加入28μL AMPure XP beads和14μL乙醇进行纯化并用10μL Nuclease Free H2O洗脱，得到加了A尾的dsDNA。
(S66)Y型Adapter连接：
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000008
将上述实验体系放入PCR仪中并在20℃下孵育20min，随后与24μL“12P XP”beads混合并在室温下孵育6min，之后保留上清液并将其与12μL AMPure XP beads和5μL 40wt％PEG8000混合，再在室温下孵育6min，随后用10μL Nuclease Free H2O洗脱两次，得到洗脱液，之后将洗脱液与12μL AMPure XP混合，再次在室温下孵育6min，然后使用30μL Qiagen EB洗脱1次，得到产物。
(S67)第二链消化和文库扩增
将15μL上述产物与1μL Uracil DNA Glycosylase混合并在37℃下孵育30min，得到UNG digested DNA。然后在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000009
将上述实验体系近PCR扩增，循环条件为：94℃30s；(98℃10s；65℃30s；72℃30s)循环15个循环；72℃5min；然后将实验体系的温度保持在4℃。随后加入43μL AMPure XP beads进行纯化并利用12μL Qiagen EB进行洗脱，得到文库。
(S68)质检及准备标引文库
先用Qubit荧光计检测文库浓度，结果为4.06ng/μL。然后使用Agilent 2100生物分析仪检测文库质量，检测结果见图2，结果显示插入片段大小合格，峰型单一，峰值在200-500bp左右。随后根据检测浓度，将不同的标引文库混合。用Qubit荧光计检测文库浓度后开始续 上机测序工作。
实施例2 新鲜肝癌组织样品中的环状RNA高通量测序文库的构建
(S1)新鲜肝癌组织样品中的总RNA的提取
取新鲜肝癌组织样品50g，在4℃下用PBS洗一次，并剪碎组织。随后加入1mL Trizol，并用手提式高速分散器匀浆15次。之后将样品收集到EP管内并加入200μL氯仿，上下颠倒混匀30s后静置3min。随后再在4℃的温度和12000rpm的转速下离心15min，溶液裂解液分三层，其中上层为溶于水相的RNA。取上层溶液并向其中加入等体积的异丙醇，混匀并静置10min。之后再次在4℃的温度和12000rpm的转速下离心10min并移除上清液。随后在沉淀中加入1mL 75％乙醇并上下颠倒混匀重悬沉淀。然后还在4℃的温度和12000rpm的转速下离心10min并移除上清，保留沉淀。然后将沉淀在室温下干燥15min直至管壁无液体。随后加入30μL DEPC H2O溶解RNA，以获得RNA溶液。
(S2)去除新鲜组织样品中的DNA
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000010
然后将上述实验体系在室温下孵育30min，之后加入300μL Buffer RP并涡旋混匀15s，随后静置10min以使DNase I失活。其后加入250μL无水乙醇并涡旋混匀15s，随后进行离心以去除管壁上的液滴。然后使用Hipure RNA柱子纯化RNA，并使用25μL DEPC H2O进行洗脱，得到去除DNA后的RNA样品。
(S3)检测并评定样品中的总RNA质量，确定RNA质量符合要求
利用1％琼脂糖凝胶电泳检测去除DNA后的RNA样品中的RNA降解程度并确定是否有污染，结果显示该样品无污染且RNA降解程度较小。随后利用紫外分光光度计检测RNA的纯度，其中OD260/280比值为2.05，纯度较高。之后再通过Agilent 2100生物分析仪分析RNA的完整性，显示RNA完整性较好，RIN为7.1。
(S4)去除rRNA
设计195条与新鲜组织中的rRNA序列互补的50bp DNA探针等质量混合在一起，形成 DNA探针库，然后配制如下所示的实验体系：
DNA探针库                  5μg
去除DNA后的RNA样品         5μg
5×hybridization buffer    5μL(其成分为1M NaCl，0.5M Tris-HCl，pH7.4)
补DEPC水至25μL。
将上述实验体系在95℃下混合两分钟，然后以0.1℃/s的速度将实验体系的温度降低至45℃，形成DNA-RNA杂交物。随后将10μL RNase H(5U/μL)和5μL已预热至37℃的10×RNase H Reaction Buffer(其成分为：500mM Tris-HCl，750mM KCL，30mM MgCl2，100mM DTT，pH＝8.3)和5μL DEPC水加入至DNA-RNA杂交物中。然后在37℃的温度下消化与DNA杂交的RNA 30min并使用2.2×RNA Clean XP beads进行纯化。之后向反应体系中加入5μL Turbo DNaseⅠ(2U/μL)并在37℃的条件下消化DNA探针30min。随后使用2.2×RNA Clean XP beads进行纯化，得到rRNA耗竭的RNA，同时用Qubit荧光计测定rRNA耗竭的RNA浓度，结果显示浓度为24.8ng/μL。
(S5)去除线性RNA
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000011
将上述体系置于37℃水浴中消化30min，获得线性RNA去除的RNA，同时用Qubit荧光计测定其浓度，结果显示浓度为8.2ng/μL。
(S6)构建用于高通量测序的环状RNA文库
(S61)RNA片段化
配制如下所示的实验体系：
5×First Strand Buffer   8μL
线性RNA去除的RNA         10μL
Nuclease free H2O        2μL；
将上述实验体系放入PCR仪中孵育，所述PCR仪的程序为：94℃5min，然后立刻置于冰上，得到片段化后的RNA。
(S62)第一链合成：
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000012
将上述实验体系放入PCR仪中孵育，所述PCR仪的程序为：65℃3min，然后置于冰上，并加入4μL Nuclease free H2O、1μL 100mM DTT、0.1μL 25mM dNTPs、0.5μL SupeRase-In、0.5μL M-MulVReverse Transcriptase和4μg Actinomycin D。随后将反应体系放入PCR仪中孵育，所述PCR仪的程序为：25℃10min；-42℃50min；-70℃15min；-4℃Hold。之后加入38μL RNA Clean XP和19μL 100％ethanol进行纯化，并使用16μL Nuclease Free H2O进行洗脱，获得RNA/cDNA杂交物，同时转移至新管中。
(S63)第二链合成
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000013
将上述实验体系在16℃下孵育2.5h。随后加入38μL RNA Clean XP和19μL乙醇进行纯化并用32μL Qiagen EB进行洗脱，得到dsDNA。
(S64)末端修复
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000014
将上述实验体系放入PCR仪中并在20℃下保持30min。随后加入28μL RNA Clean XP 和14μL乙醇进行并用17μL Nuclease Free H2O进行洗脱，得到末端修复后的dsDNA。
(S65)加A尾
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000015
然后在37℃下孵育30min。随后加入28μL AMPure XP beads和14μL乙醇进行纯化并用10μL Nuclease Free H2O洗脱，得到加了A尾的dsDNA。
(S66)Y型Adapter连接：
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000016
将上述实验体系放入PCR仪中并在20℃下孵育20min，随后与24μL“12P XP”beads混合并在室温下孵育6min，之后保留上清液并将其与12μL AMPure XP beads和5μL 40wt％PEG8000混合，再在室温下孵育6min，随后用10μL Nuclease Free H2O洗脱两次，得到洗脱液，之后将洗脱液与12μL AMPure XP混合，再次在室温下孵育6min，然后使用30μL Qiagen EB洗脱1次，得到产物。
(S67)第二链消化和文库扩增
将15μL上述产物与1μL Uracil DNA Glycosylase混合并在37℃下孵育30min，得到UNG digested DNA。然后在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000017
将上述实验体系近PCR扩增，循环条件为：94℃30s；(-98℃10s；-65℃30s；-72℃ 30s)循环15个循环；-72℃5min；然后将实验体系的温度保持在-4℃。随后加入43μL AMPure XP beads进行纯化并利用12μL Qiagen EB进行洗脱，得到文库。
(S68)质检及准备标引文库
先用Qubit荧光计检测文库浓度，结果为5.16。然后使用Agilent 2100生物分析仪检测文库质量，检测结果见图2，插入片段大小合格，峰型单一，峰值在200-500bp左右。随后根据检测浓度，将不同的标引文库混合。用Qubit荧光计检测文库浓度后开始上机测序工作。
实施例3 FFPE样品中的环状RNA高通量测序文库的构建
(S1)FFPE样品中的总RNA的提取
取FFPE样品切片成8μm厚的片状，立即转移7片切片至1.5mL离心管。然后加入1mL二甲苯并剧烈涡旋30s，并在14000rpm的转速下离心2min。之后加入1mL乙醇至样品中并涡旋混匀30s，再在14000rpm的转速下离心2min，移除上清，保留沉淀。在37℃的温度下干燥15min以去除乙醇。随后向沉淀中加入200μL裂解液和20μl Proteinase K并涡旋混匀。其后在55℃的温度下水浴15min，之后在80℃的温度下水浴15min。低速短暂离心后加入200μL缓冲液至样品中并涡旋混匀20s。随后加入600μL无水乙醇至样品中并涡旋混匀20s。之后转移混合液至吸附柱中，并在8000rpm的转速下离心50s，倒弃流出液，把柱子装在收集管中。随后加入500μL漂洗液至柱子中，再在8000rpm的转速下离心50s，倒弃流出液，把柱子装在收集管中。其后再加入500μL漂洗液至柱子中，再在8000rpm的转速下离心50s。倒弃流出液，把柱子装在收集管中。随后在13000rpm的转速下离心3min，以甩干柱子的基质，并将柱子转移至新的1.5mL离心管中。随后加入30μL DEPC水至柱子的膜中央并静置2min，然后在13000rpm的转速下离心1min以获得RNA溶液。
(S2)去除FFPE样品中的DNA
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000018
然后将上述实验体系在室温下孵育30min，之后加入300μL Buffer RP并涡旋混匀15s，随后静置10min以使DNase I失活。其后加入250μL无水乙醇并涡旋混匀15s，随后进行 离心以去除管壁上的液滴。然后使用Hipure RNA柱子纯化RNA，并使用25μL DEPC H2O进行洗脱，得到去除DNA后的RNA样品。
(S3)检测并评定样品中的总RNA质量，确定RNA质量符合要求
利用1％琼脂糖凝胶电泳检测去除DNA后的RNA样品中的RNA降解程度并确定是否有污染，结果显示该样品RNA明显降解。随后利用紫外分光光度计检测RNA的纯度，其中OD260/280比值为2.37。之后再通过Agilent 2100生物分析仪分析RNA的完整性，显示RNA完整性很差，RIN值为4.5。
(S4)去除rRNA
设计195条与FFPE样本中的rRNA序列互补的50bp DNA探针等质量混合在一起，形成DNA探针库，然后配制如下所示的实验体系：
DNA探针库                  5μg
去除DNA后的RNA样品         5μg
5×hybridization buffer    5μL(其成分为1M NaCl，0.5M Tris-HCl，pH 7.4)
补DEPC水至25μL。
将上述实验体系在95℃下混合两分钟，然后以0.1℃/s的速度将实验体系的温度降低至45℃，形成DNA-RNA杂交物。随后将10μL RNase H(5U/μL)和5μL已预热至37℃的10×RNase H Reaction Buffer(其成分为：500mM Tris-HCl，750mM KCL，30mM MgCl2，100mM DTT，pH＝8.3)和5μL DEPC水加入至DNA-RNA杂交物中。然后在37℃的温度下消化与DNA杂交的RNA 30min并使用2.2×RNA Clean XP beads进行纯化。之后向反应体系中加入5μL Turbo DNaseⅠ(2U/μL)并在37℃的条件下消化DNA探针30min。随后使用2.2×RNA Clean XP beads进行纯化，得到rRNA耗竭的RNA，同时用Qubit荧光计测定rRNA耗竭的RNA浓度，结果显示浓度为19.9ng/μL。
(S5)去除线性RNA
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000019
将上述体系置于37℃水浴中消化30min，获得线性RNA去除的RNA，同时用Qubit 荧光计测定其浓度，结果显示浓度为3.25ng/μL。
(S6)构建用于高通量测序的环状RNA文库
(S61)RNA片段化
配制如下所示的实验体系：
5×First Strand Buffer      8μL
线性RNA去除的RNA            10μL
Nuclease free H2O           2μL；
将上述实验体系放入PCR仪中孵育，所述PCR仪的程序为：94℃5min，然后立刻置于冰上，得到片段化后的RNA。
(S62)第一链合成：
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000020
将上述实验体系放入PCR仪中孵育，所述PCR仪的程序为：65℃3min，然后置于冰上，并加入4μL Nuclease free H2O、1μL 100mM DTT、0.1μL 25mM dNTPs、0.5μL SupeRase-In、0.5μL M-MulVReverse Transcriptase和4μg Actinomycin D。随后将反应体系放入PCR仪中孵育，所述PCR仪的程序为：25℃10min；-42℃50min；-70℃15min；-4℃Hold。之后加入38μL RNA Clean XP和19μL 100％ethanol进行纯化，并使用16μL Nuclease Free H2O进行洗脱，获得RNA/cDNA杂交物，同时转移至新管中，。
(S63)第二链合成
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000021
将上述实验体系在16℃下孵育2.5h。随后加入38μL RNA Clean XP和19μL乙醇进行纯化并用32μL Qiagen EB进行洗脱，得到dsDNA。
(S64)末端修复
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000022
将上述实验体系放入PCR仪中并在20℃下保持30min。随后加入28μL RNA Clean XP和14μL乙醇进行并用17μL Nuclease Free H2O进行洗脱，得到末端修复后的dsDNA。
(S65)加A尾
配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000023
然后在37℃下孵育30min。随后加入28μL AMPure XP beads和14μL乙醇进行纯化并用10μL Nuclease Free H2O洗脱，得到加了A尾的dsDNA。
(S66)Y型Adapter连接：
在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000024
将上述实验体系放入PCR仪中并在20℃下孵育20min，随后与24μL“12P XP”beads混合并在室温下孵育6min，之后保留上清液并将其与12μL AMPure XP beads和5μL 40wt％PEG8000混合，再在室温下孵育6min，随后用10μL Nuclease Free H2O洗脱两次，得到洗脱液，之后将洗脱液与12μL AMPure XP混合，再次在室温下孵育6min，然后使用30μL Qiagen EB洗脱1次，得到产物。
(S67)第二链消化和文库扩增
将15μL上述产物与1μL Uracil DNA Glycosylase混合并在37℃下孵育30min，得到UNG digested DNA。然后在0℃下配制如下所示的实验体系：
Figure PCTCN2017093865-appb-000025
将上述实验体系近PCR扩增，循环条件为：94℃30s；(98℃10s；65℃30s；72℃30s)循环15个循环；72℃5min；然后将实验体系的温度保持在4℃。随后加入43μL AMPure XP beads进行纯化并利用12μL Qiagen EB进行洗脱，得到文库。
(S68)质检及准备标引文库
先用Qubit荧光计检测文库浓度，结果为5.24ng/μL。然后使用Agilent 2100生物分析仪检测文库质量，检测结果见图2，插入片段大小合格，峰型单一，峰值在200-500bp左右。随后根据检测浓度，将不同的标引文库混合。用Qubit荧光计检测文库浓度后开始续上机测序工作。
测序实施例生物信息学分析测序数据
将实施例1-3构建的环状RNA高通量测序文库及文献报道方法(参见Molecular Cell 56,55–66,October 2,2014)构建的环状RNA高通量测序文库在Illumina测序平台进行测序。结果示于表1中。
表1本发明方法与文献报道方法测序结果生物信息学分析
Figure PCTCN2017093865-appb-000026
测序数据经生物信息学分析之后的结果见表1，对比之下，本发明实施例提供的方法能够将样品中的rRNA进一步去除，数据中rRNA残留比例<0.1％，而文献报道方法中rRNA残留为5％，对于FFPE等降解严重的RNA样品更是高达37％，严重浪费数据量，本发明方法使得环状RNA的有效测序数据量显著提高。在环状RNA检测数量方面，RNA完整性好的样品(细胞样品)用文献报道方法检出的环状RNA较多，但是RNA质量差的样品，尤其是FFPE样品本发明方法显著优于文献报道方法。此外，使用本发明方法进行环状RNA高通量测序文库构建，有标准的操作流程，条件优化，结果稳定，成本仅为文献报道方法的1/3。从测序结果中随机挑选20个环状RNA进行QPCR验证，验证结果阳性率100％(图3)，有力证实了本发明方法的可靠性。
由此可证明本发明实施例所用的方法能显著的将样品中的环状RNA 进一步富集，大幅度降低环状RNA 测序数据量，降低成本，减少人力物力的浪费。
根据上述说明书的揭示和教导，本发明所属领域的技术人员还可以对上述实施方式进行变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对发明的一些修改和变更也应当落入本发明的权利要求的保护范围内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制。

Claims (9)

  1. 一种环状RNA高通量测序文库的构建方法，其依次包括以下步骤：
    (S1)提取样品中的总RNA；
    (S2)去除样品中的DNA；
    (S3)检测并评定样品中的总RNA质量，确定RNA质量符合要求；
    (S4)去除rRNA；
    (S5)去除线性RNA；
    (S6)构建用于高通量测序的环状RNA文库。
  2. 根据权利要求1所述的构建方法，其特征在于，所述步骤(S3)中检测样品中总的RNA质量依次包括以下步骤：
    (S31)利用1％琼脂糖凝胶电泳检测RNA降解程度以及是否有污染；
    (S32)利用紫外分光光度计检测RNA的纯度，即OD260/280比值；
    (S33)检测RNA的完整性。
  3. 根据权利要求1所述的构建方法，其特征在于，所述步骤(S4)去除rRNA依次包括以下步骤：
    (S41)将与rRNA序列互补的DNA探针等质量混合，形成DNA探针库；
    (S42)将所获得DNA探针库与经过检测并评定样品中的总RNA质量确定RNA质量符合要求的RNA混合杂交，形成DNA-RNA杂交物；
    (S43)用RNase H消化所获得的DNA-RNA杂交物；
    (S44)用DNase I消化DNA探针，获得rRNA耗竭的RNA；
    (S45)通过荧光计测定rRNA耗竭的RNA的浓度。
  4. 根据权利要求3所述的构建方法，其特征在于，所述步骤(S42)中所用的DNA探针库与RNA的重量比为(1-2)：1，优选1:1。
  5. 根据权利要求3所述的构建方法，其特征在于，所述步骤(S42)中混合杂交所采用的条件为在95℃的温度下杂交2分钟，然后以0.1℃/s的速度将温度缓慢地降低至45℃。
  6. 根据权利要求3所述的构建方法，其特征在于，所述步骤(S43)中用RNase H消化所获得的DNA-RNA杂交物所采用的条件为在37℃的温度下消化30分钟。
  7. 根据权利要求1所述的构建方法，其特征在于，所述步骤(S6)中构建用于高通量测序的环状RNA文库依次包括以下步骤：
    (S61)RNA片段化；
    (S62)第一链合成：采用逆转录酶加随机引物的方法进行；
    (S63)第二链合成：利用RNase H消化RNA/cDNA杂交物中的RNA链，然后通过DNA聚合酶I合成第二条链；
    (S64)末端修复：利用End Repair Mix形成平末端的dsDNA并在5'端加上磷酸基团；
    (S65)加A尾：利用Klenow 3'-5'exo-在dsDNA末端加A尾；
    (S66)Y型Adapter连接：利用T4 DNA Ligase连接Adapter和加了A尾的dsDNA；
    (S67)第二链消化和文库扩增：利用UNG酶消化第二链；
    (S68)质检及准备标引文库。
  8. 根据权利要求7所述的构建方法，其特征在于，所述步骤(S61)中RNA片段化的条件为在90-96℃，优选94℃的温度下片段化3-6分钟，优选5分钟。
  9. 一种环状RNA高通量测序文库构建的试剂盒，其特征在于，其包括：
    (1)环状RNA富集试剂、DNase I、RNase H、RNaseR；
    (2)种属特异的DNA探针；
    (3)文库构建所需的各种酶：DNA聚合酶I、Large Fragment、T4 Polynucleotide Kinase、Klenow Fragment、Escherichia coli UDG、T4 DNA Ligase、Phusion High-Fidelity DNA Polymerase；
    (4)磁珠、dNTP、Nuclease Free Water、dUTP、接头、引物，
    其中，所述种属特异的DNA探针能与相应物种的rRNA序列互补。
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