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WO2018149091A1 - Procédé de construction d'une bibliothèque de séquençage à haut débit d'arn circulaire et kit associé - Google Patents

Procédé de construction d'une bibliothèque de séquençage à haut débit d'arn circulaire et kit associé 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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Chinese (zh)
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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

L'invention concerne un procédé de construction d'une bibliothèque de séquençage à haut débit d'ARN circulaire et un kit associé. Le procédé de construction comprend séquentiellement les étapes consistant à : (S1) extraire l'ARN total d'un échantillon; (S2) éliminer l'ADN de l'échantillon; (S3) détecter et évaluer la qualité de l'ARN total dans l'échantillon et déterminer si la qualité de l'ARN satisfait aux exigences; (S4) éliminer l'ARNr; (S5) éliminer l'ARN linéaire; et (S6) construire la bibliothèque d'ARN circulaire pour séquençage à haut débit. Le procédé de construction de la bibliothèque de séquençage à haut débit d'ARN circulaire offre une efficacité et une stabilité élevées; il présente un faible taux résiduel d'ARNr; il offre une efficacité élevée de détection d'ARN circulaire et une bonne répétabilité des données; et il présente un taux de réussite de vérification élevé pour les résultats de séquençage. Ce procédé peut en particulier être appliqué à un tissu fixé au formol et enrobé dans de la paraffine (FFPE) et d'autres échantillons à faible qualité d'ARN.
PCT/CN2017/093865 2017-02-20 2017-07-21 Procédé de construction d'une bibliothèque de séquençage à haut débit d'arn circulaire et kit associé Ceased WO2018149091A1 (fr)

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