WO2012109963A1 - Rna cleaving agent and use thereof - Google Patents

Rna cleaving agent and use thereof Download PDF

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WO2012109963A1
WO2012109963A1 PCT/CN2012/070962 CN2012070962W WO2012109963A1 WO 2012109963 A1 WO2012109963 A1 WO 2012109963A1 CN 2012070962 W CN2012070962 W CN 2012070962W WO 2012109963 A1 WO2012109963 A1 WO 2012109963A1
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rna
fragmentation reagent
sequencing
concentration
reagent according
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PCT/CN2012/070962
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French (fr)
Chinese (zh)
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章文蔚
陈海燕
胡轶霖
田方
张艳艳
张秀清
杨焕明
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深圳华大基因科技有限公司
深圳华大基因研究院
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Publication of WO2012109963A1 publication Critical patent/WO2012109963A1/en

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    • 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

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  • the present invention relates to the field of second generation high throughput sequencing, particularly in the field of RNA sequencing.
  • the invention relates to RNA fragmentation reagents and uses thereof. More specifically, the present invention provides an RNA fragmentation reagent, a method of interrupting RNA, a method of constructing an RNA sequencing library, an RNA sequencing library, and a sequencing method. Background technique
  • RNA sequencing has become an important new tool for gene expression and transcriptome analysis.
  • a transcriptome is the sum of all RNAs that a particular cell can transcribe under a certain functional state, including mRNA and non-coding RNA.
  • RNA-Seq include, first, the use of Poly(T) oligonucleotides to extract all RNAs with Poly(A) tails from total RNA, mainly encoding the mRNA transcribed from the gene, and then the resulting The RNA is randomly broken into fragments, the RNA fragments are recovered by ethanol precipitation, and the cDNA fragments are synthesized from the RNA fragments by random primers and reverse transcriptase. Then, the cDNA fragments are end-repaired and ligated to the sequencing adaptor, which will be used for sequencing. cDNA. In this process, RNA was selected to be interrupted first, rather than breaking cDNA after cDNA synthesis because the RNA disruption was relatively simple and fast, and the randomness and uniformity of the break were good.
  • RNA fragmentation reagents are commonly used to fragment RNA.
  • the steps are cumbersome, complicated, and the experiment efficiency is low, which is not suitable for the operation of batch samples, which is not conducive to the automation of the entire method.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides RNA fragmentation reagents and uses thereof to overcome the deficiencies of commercially available RNA fragmentation reagents.
  • the invention provides an RNA fragmentation reagent. According to an embodiment of the invention, the
  • the RNA fragmentation reagent comprises: a biological buffer, wherein the biological buffer is suitable for maintaining the pH of the RNA fragmentation reagent between 7 and 9; a monovalent metal ion and a divalent metal ion, wherein the biological buffer, one Both the valence metal ion and the divalent metal ion are at an effective concentration.
  • the type of the biological buffer is not particularly limited.
  • the term "biological buffer” as used in the present invention refers to a liquid which is capable of stabilizing the pH of a reaction system, that is, it is capable of timely adjusting the pH in a reaction system in which a biological reaction is involved, and the value of the pH is made. Maintain the special required for the reaction Within a certain range, the reaction can proceed smoothly.
  • the biological buffer can be a Tris buffer.
  • the Tris buffer maintains the pH of the RNA fragmentation reagent between 7 and 9.
  • the concentration of the Tris buffer is not particularly limited as long as the action of the Tris buffer can be achieved, that is, the pH of the reaction system can be stabilized, and the pH of the RNA fragmentation reagent of the present invention is maintained at 7 to 9.
  • the concentration of the Tris buffer may be from 100 to 350 mM.
  • the concentration of the Tris buffer may be from 150 to 300 mM.
  • the concentration of the Tris buffer may be from 200 to 250 mM.
  • the monovalent metal ion in the RNA fragmentation reagent of the present invention, may be at least one of K+ and Na + . According to a specific example of the invention, the monovalent metal ion is K + . According to an embodiment of the present invention, the concentration of the monovalent metal ion may be 80 to 400 mM. According to a specific example of the present invention, the concentration of the monovalent metal ion may be from 90 to 300 mM. According to some embodiments of the invention, the concentration of monovalent metal ions may range from 100 to 250 mM. According to an embodiment of the present invention, the divalent metal ion may be at least one of Mg 2+ and Zn 2+ .
  • the source of RNA is not particularly limited.
  • the RNA may be derived from a prokaryotic or eukaryotic organism.
  • the expression "RNA may be derived from a prokaryotic or eukaryotic organism" as used herein means that the RNA fragmentation reagent according to an embodiment of the present invention is suitable for cleavage of RNA derived from prokaryotes and eukaryotes, thereby RNA can be effectively applied to the construction of high-throughput RNA sequencing libraries.
  • the term "effective concentration" as used herein means that the concentration of a biological buffer, a metal ion including a monovalent metal ion and a divalent metal ion enables its corresponding function in a related process, for example, a biological
  • concentration of the buffer is sufficient to maintain the pH of the reagent within a desired range, for example, 7-9, the concentration of the valence metal ion can reach the concentration required for the synthesis of the first strand of the cDNA, and the concentration of the divalent metal ion can achieve the interruption.
  • the concentration required for RNA is sufficient to maintain the pH of the reagent within a desired range, for example, 7-9, the concentration of the valence metal ion can reach the concentration required for the synthesis of the first strand of the cDNA, and the concentration of the divalent metal ion can achieve the interruption.
  • concentration required for RNA is the concentration required for RNA.
  • the RNA fragmentation reagent is used for RNA fragmentation, and the breaking effect is equivalent to the RNA Fragmentation Reagents (AM8740) provided by ABI, but the reagent is utilized by adjusting the breaking temperature and the breaking time.
  • the RNA can be interrupted into fragments of different lengths, and the RNA fragmentation reagent of the present invention is used for RNA fragmentation, and the steps are simple, convenient, less time-consuming, low in cost, and reproducible.
  • the inventors have surprisingly found that RNA fragmentation reagents according to embodiments of the present invention can be effectively applied to RNA sequencing library construction of high throughput sequencing platforms and are suitable for RNA sequencing library construction of large samples.
  • the RNA fragmentation reagent of the present invention may comprise: a biological buffer that maintains a pH between 7 and 9, a monovalent metal ion and a divalent metal ion; wherein the biological buffer, Both the monovalent metal ion and the divalent metal ion are at an effective concentration.
  • monovalent metal ions are necessary for the synthesis of the first strand of cDNA, and divalent metal ions are necessary for interrupting RNA.
  • the biological buffer may be a Tris buffer at a concentration of 100-350 mM, preferably 150-300 mM, more preferably 200-250 mM; maintaining the pH of the RNA fragmentation reagent between 7 and 9 .
  • the monovalent metal ion may be K+ and/or Na + , preferably K+; the valence metal ion concentration may be 80-400 mM, preferably 90-300 mM, more preferably 100-250 mM.
  • the divalent metal cation may be Mg 2+ and/or Zn 2+ , preferably Mg 2+ and Zn 2+ ; the concentration of Mg 2+ ions may be 8-100 mM, preferably 10-80 mM. More preferably, it is 15-60 mM; the concentration of Zn 2+ ions may be 8-100 mM, preferably 10-80 mM, more preferably 15-60 mM.
  • RNA in the RNA fragmentation reagent of the present invention, may be derived from prokaryotic or eukaryotic organisms, and may be derived from a single cell or a plurality of cells, a single cell or a plurality of cells, a single tissue or Multiple tissues, single tissue or multiple tissues, one organism or multiple organisms, one organism or multiple organisms.
  • RNA fragmentation reagent according to the embodiment of the present invention and the RNA Fragmentation Reagents (AM8740) provided by ABI Company have the same breaking effect, and can be satisfied by adjusting the breaking temperature and the breaking time. The size of the fragment needs.
  • the RNA cleavage reagent is applied to the construction of an RNA sequencing library, and the other advantage is that: after cleavage of the RNA, no need to add a reaction terminator, and without any purification step, the subsequent reverse transcription reaction can be directly performed, that is, In a specific reverse transcription reaction system, the cleavage reagent can replace the first strand synthesis buffer without additional addition of the first strand synthesis buffer, and the obtained data is based on the standard procedure for preparation of the Illumina mRNA-Seq library (see mRNA The data obtained by Sequencing Sample Preparation Guide, part #l 004898, which is incorporated herein by reference in its entirety, is consistent, which indicates that the method of constructing an RNA sequencing library using the RNA fragmentation reagent simplifies the operation steps, saving time and reducing costs. And on the basis of not affecting the results, it creates very favorable conditions for automated operation, making it possible to build automated batch RNA samples.
  • the present invention provides the use of an RNA fragmentation reagent for interrupting RNA according to an embodiment of the present invention.
  • the invention provides a method of disrupting RNA.
  • the method comprises: disrupting RNA using an RNA fragmentation reagent according to an embodiment of the invention.
  • the method for interrupting RNA according to an embodiment of the present invention can easily, quickly and efficiently interrupt RNA of prokaryotic and eukaryotic organisms, and has low cost, good reproducibility, and RNA fragments obtained after interruption. Can be effectively applied to the construction of RNA sequencing libraries.
  • the present invention provides a method of constructing an RNA sequencing library. According to an embodiment of the invention, the method may comprise the following steps:
  • RNA is disrupted using an RNA cleavage reagent according to an embodiment of the invention to obtain an RNA fragment.
  • RNA is interrupted using an RNA cleavage reagent at a temperature of 70-100 °C.
  • the RNA is interrupted using the RNA fragmentation reagent at a temperature of 85-95 °C.
  • the RNA is interrupted using the RNA fragmentation reagent at a temperature of 94 °C.
  • the duration of action of the RNA treatment using the RNA fragmentation reagent is 3-12 min.
  • the duration of action of the RNA treatment using the RNA fragmentation reagent is 6-10 min. According to one embodiment of the invention, the duration of action of the RNA treatment using the RNA fragmentation reagent is 10 min. According to an embodiment of the invention, the length of the RNA fragment is between 60 and 1500 nt. According to a specific example of the invention, the length of the RNA fragment is between 150 and 700 nt. According to some embodiments of the invention, the RNA fragments are 150-500 nt in length. (b) Reverse transcriptase and random primers are directly added to the reaction system after the step (a) to perform reverse transcription to synthesize the first strand of cDNA.
  • step (c) adding a polymerase and a cDNA double-strand synthesis buffer to the reaction system after the step (b) to synthesize the second strand of the cDNA and obtain a double-stranded cDNA.
  • Klenow which is an exonuclease activity, adds a base A to the 3' end of the double-stranded cDNA which is end-repaired to obtain a double-stranded cDNA having a sticky terminal A, which can then be viscous with a sticky end A using T4 DNA ligase.
  • the double-stranded cDNA is ligated to the sequencing link, whereby the ligation product can be efficiently obtained.
  • the method for constructing an RNA sequencing library of the present invention can be used for large-scale, automated, industrialization because of simple steps, easy operation, and low cost, and is very suitable for RNA sequencing library construction of large-scale samples. produce.
  • the method for constructing an RNA sequencing library of the present invention may further comprise the following steps:
  • step (b) directly adding a reverse transcriptase and a random primer to the reaction system after the step (a), and performing reverse transcription to synthesize the first strand of the cDNA;
  • step (c) adding a polymerase and a cDNA double-strand synthesis buffer to the reaction system after the step (b) to synthesize a second strand of the cDNA to obtain a double-stranded cDNA;
  • step (d) purifying the double-stranded cDNA obtained in step (c);
  • step (f) PCR-amplifying the ligation product obtained in step (e) to obtain an RNA sequencing library.
  • the action temperature at which RNA is interrupted by the RNA fragmentation reagent may be 70 to 100 ° C, preferably 85 to 95 ° C, more preferably 94 ° C.
  • the duration of action of RNA disruption using an RNA cleavage reagent can be from 3 to 12 min, preferably from 6 to 10 min, more preferably 10 min.
  • the invention provides an RNA sequencing library.
  • the RNA sequencing library is constructed by the method of the invention for constructing an RNA sequencing library. The inventors have found that an RNA sequencing library according to an embodiment of the present invention can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and based on the sequencing result, can accurately and efficiently determine the RNA sequence information of the sample, thereby based on the obtained information. , can effectively carry out gene expression and transcriptome research on the sample.
  • the invention provides a sequencing method.
  • the method may comprise the steps of: constructing an RNA sequencing library according to the method of constructing an RNA sequencing library according to an embodiment of the present invention; and sequencing the RNA sequencing library, wherein the sequencing uses a high-throughput sequencing platform Performed, the high throughput sequencing platform is at least one selected from the group consisting of Illumina/Solexa, ABI Solid, and Roche 454 sequencing platforms.
  • the sequencing method of the present invention can conveniently and efficiently construct an RNA sequencing library of a sample, and can accurately sequence the RNA sequencing library, and the method is simple, easy to operate, less time-consuming, and cost-effective. Low, repeatable, and accurate and reliable sequencing results, the method can be applied to RNA sequencing of a large number of samples, and can be applied to large-scale industrial automation production.
  • the sequencing method of the present invention may further comprise the step of constructing an RNA sequencing library according to an embodiment of the present invention and sequencing the sequencing library.
  • sequencing can be performed by any sequencing method, including but not limited to the dideoxy chain termination method; preferably a high throughput sequencing method, including but not limited to a second generation sequencing platform or a single molecule sequencing platform .
  • the second generation sequencing platform see Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet.
  • RNA fragmentation reagent of the present invention and its use are completed by the inventor of the present application through arduous creative labor and optimization work.
  • the additional aspects and advantages of the invention will be set forth in part in the description which follows.
  • FIG. 12 Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3xRNA cleavage reagent in Example 11, wherein the RNA fragment is from a parallel sample of maize RNA;
  • Figure 13 shows the use in Example 11 Agilent Bioanalyzer 2100 assay of the size of the RNA fragment obtained by cleavage of the 3xRNA fragmentation reagent at 94 ° C for 10 min, wherein the RNA fragment is derived from rice RNA;
  • Figure 14 Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained in Example 1 using the 3xRNA cleavage reagent at 94 °C for 10 min, in which the RNA fragment was derived from a parallel sample of rice RNA;
  • Figure 15 Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3x RNA fragmentation reagent in Example 11, wherein the RNA fragment is derived from fungal RNA;
  • 5xRNA cleavage buffer (Applied Biosystem); the source of other materials, reagents or buffers is the same as step 1.1 in Example 1.
  • Table 1 shows the reads of the same sample, a and b.
  • the ratio is equivalent, and the positive and negative deviations are less than 5%. It can be considered that there is no significant difference between the two methods.
  • Table 2 shows the correlation analysis between the gene expression amount and the QPCR detection results in the sequencing data obtained by the two methods of database construction a and b. Specifically, the correlation analysis results are obtained by performing Spearman correlation analysis on the gene expression amount obtained by two different database construction methods and the same sample obtained by QPCR detection, wherein the correlation coefficient is from 0-1, and the number is larger, indicating The closer the two are, the better the correlation.
  • Table 3 shows that for the same sample, the number of genes detected by the same data amount is similar for the two different database construction methods, thus confirming that the database construction method b of the present invention is consistent with the data obtained by the method a provided by Illumina.
  • the RNA fragmentation reagent of the present invention and its breaking conditions are suitable for mRNA-seq construction, and the obtained data is true and reliable, and has no influence on information analysis.
  • Table 2 Correlation analysis between gene expression levels of MAQC standard library constructed by a and b methods and QPCR detection results
  • RNA sample MAQC-AB or MAQC-Agilent
  • MAQC-AB or MAQC-Agilent was used to detect the RNA fragmentation reagent component and concentration with a fixed breaking time of 10 min and an interrupting temperature of 94 °C.
  • RNA fragmentation reagent 350 mM Tris-HCl (H 8.0 ), 80 mM NaCl, 100 mM MgCl 2 , 8 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); other sample and reagent buffers
  • the source is the same as in Example 1.
  • RNA fragmentation reagent 150 mM Tris-HCl (pH 8.0), 300 mM KC1, 90 mM NaCl, 80 mM MgCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); source of other sample and reagent buffers Same as Example 1.
  • RNA fragmentation reagent 300 mM Tris-HCl (pH 8.0), 250 mM KC1, 100 mM NaCl, 10 mM ZnCl 2
  • the components of the fragmentation reagent are all from Sigma and the reagents are free of RNase); the source of the other sample and reagent buffers is the same as in Example 1.
  • RNA fragmentation reagent 250 mM Tris-HCl (pH 8.0), 100 mM KC1, 100 mM NaCl, 60 mM MgCl 2 ,
  • RNA fragmentation reagent 200 mM Tris-HCl (pH 8.0), 90 mM NaCl, 100 mM MgCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); the source of other sample and reagent buffers is the same as the example 1.
  • 3xRNA cleavage reagent lOOmM Tris-HCl (pH 8.0), 250 mM NaCl, 8 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent does not contain RNase); the source of other sample and reagent buffer is the same as the example 1.
  • 3xRNA cleavage reagent 200 mM Tris-HCl (H 8.0 ), 300 mM KC1, 80 mM MgCl 2 (the components of the fragmentation reagent were from Sigma, the reagent contained no RNase); the source of the other sample and reagent buffer was the same as in Example 1.
  • 3xRNA cleavage reagent 200 mM Tris-HCl (pH 8.0), 100 mM KC1, 15 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); the source of other sample and reagent buffer is the same as in Example 1 .
  • the experimental procedure is the same as step 1.2.1 1.2.7 in Example 1.
  • the RNA-Seq library (MAQC-AB or MAQC-Agilent) obtained in Examples 2 to 10 was subjected to Agilent Bioanalyzer 2100 detection.
  • the test pattern shows that for the same sample, under the same rupture condition of 94 °C lOmin, the composition of the 9 components and concentration adjustment of the 3xRNA cleavage reagent is not as good as the RNA interrupting effect or with the examples.
  • the effect of 1 3x RNA fragmentation reagent 200 mM Tris-HCl (pH 8.0), 100 mM KC1, 15 mM MgCl 2 , 15 mM ZnCl 2 ) was similar.
  • the monovalent metal ion for the first strand synthesis and the divalent metal ion for the cleavage RNA are three groups.
  • the basic purpose of the present invention can be attained by mixing together and blending the three components at an effective concentration.
  • the biological buffer is preferably a Tris buffer at a concentration of 100-350 mM, preferably 150-300 mM, more preferably 200-250 mM, and maintaining the pH of the RNA fragmentation reagent between 7 and 9;
  • the monovalent metal ion is preferably K+ and/or Na + , more preferably K+, and the concentration of the valence metal ion is 80-400 mM, preferably 100- 300 mM, more preferably 200-250 mM.
  • the divalent metal cation is preferably Mg 2+ and/or Zn 2+ , more preferably Mg 2+ and Zn 2+ , wherein the concentration of Mg 2+ ions is 8-100 mM, preferably 10-80 mM, more preferably 15-60 mM, Zn
  • concentration of 2+ ions is 8-100 mM, preferably 10-80 mM, more preferably 15-60 mM.
  • RNA cleavage reagent 200 mM Tris-HCl ( ⁇ 8.0), 100 mM KC1, 15 mM MgCl 2 , 15 mM ZnCl 2 , 94 ° C lOmin
  • MAQC-Agilent and MAQC-AB RNAs from mice, rice, maize or fungi extracted with TRIzol (Invitrogen) were selected for parallel construction.
  • RNA fragments in Figures 7-18 were measured by Agilent Bioanalyzer 2100, and the results are shown in Figures 7-18.
  • Figure 7-18 shows the Agilent Bioanalyzer 2100 test chart for the size of each sample and its parallel sample RNA fragments obtained in this example.
  • the RNA fragments in Figures 7 and 8 are from MAQC-AB and their parallel samples, respectively;
  • the RNA fragments in Figures 9 and 10 are from MAQC-Agilent and their parallel samples, respectively;
  • the RNA fragments in Figure 11 and Figure 12 are from corn, respectively.
  • RNA and its parallel samples are from rice RNA and their parallel samples, respectively; the RNA fragments of Figure 15 and Figure 16 are from the fungal RN A and their parallel samples, respectively; the RNA fragments of Figure 17 and Figure 18, respectively From mouse RNA and its parallel samples. From the two repetitions of each set of samples, the RNA fragmentation reagent of the present invention and its breaking conditions are very reproducible to the same sample, and the interrupted fragments are very similar to the concentration, as shown in Fig. 9 and Fig. 10, The MAQC-Agilent and its parallel-like interrupted fragments are very close, and the peak maps are very similar.
  • the RNA fragmentation reagent of the present invention and the method for interrupting RNA have good repeatability to the same sample; between different samples, due to the particularity of the sample, the size of the fragment is slightly different, but the peak shape is It is normal, the fragment concentration is high, and the coverage is very close. Therefore, the RNA fragmentation reagent and the method for interrupting RNA of the present invention are suitable for the sample to be tested, have good breaking effect, and have good repeatability and stability. Sex. Industrial applicability
  • RNA fragmentation reagent the method for interrupting RNA
  • the method for constructing an RNA sequencing library the RNA sequencing library and the sequencing method of the invention can be effectively applied to the construction and sequencing of the RNA sequencing library of the sample, and the obtained library is of good quality and sequenced. The result is accurate.

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Abstract

Provided are an RNA cleaving agent, a method for cleaving RNA, a method for constructing an RNA sequencing library, an RNA sequencing library and a sequencing method. The RNA cleaving agent comprises: a biological buffer suitable for maintaining the pH value of the RNA cleaving agent in the range of 7 to 9; and monovalent and divalent metal ions, the biological buffer and the monovalent and divalent metal ions are in effective concentrations.

Description

RNA断裂试剂及其用途 优先权信息  RNA fragmentation reagents and their use
本申请请求 2011 年 2 月 17 日向中国国家知识产权局提交的、 专利申请号为 201110040036.9的专利申请的优先权和权益， 并且通过参照将其全文并入此处。 技术领域  Priority is claimed on Japanese Patent Application No. 201110040036.9, the entire disclosure of which is hereby incorporated by reference. Technical field
本发明涉及第二代高通量测序领域， 特别是 RNA测序领域。 具体地， 本发明涉及 RNA断裂试剂及其用途。 更具体地， 本发明提供了一种 RNA断裂试剂、 一种打断 RNA 的方法、 一种构建 RNA测序文库的方法、 一种 RNA测序文库以及一种测序方法。 背景技术  The present invention relates to the field of second generation high throughput sequencing, particularly in the field of RNA sequencing. In particular, the invention relates to RNA fragmentation reagents and uses thereof. More specifically, the present invention provides an RNA fragmentation reagent, a method of interrupting RNA, a method of constructing an RNA sequencing library, an RNA sequencing library, and a sequencing method. Background technique
随着新一代高通量 DNA测序技术的快速发展， RNA测序（RNA-seq)已成为基因表 达和转录组分析的重要新手段。转录组是特定细胞在某一功能状态下所能转录出来的所 有 RNA的总和， 包括 mRNA和非编码 RNA。 目前 RNA-Seq的主要的流程包括， 首先， 用 Poly(T)寡聚核苷酸从总 RNA中抽取全部带 Poly(A)尾的 RNA, 其中主要是编码基因 所转录的 mRNA, 然后将所得 RNA随机打断成片段， 乙醇沉淀回收 RNA片段， 再用 随机引物和逆转录酶从 RNA片段合成 cDNA片段， 然后， 对 cDNA片段进行末端修复 并连接测序接头（adapter) , 得到将用于测序的 cDNA。 在这一过程中， 之所以选择先将 RNA打断， 而不是在 cDNA合成后打断 cDNA,是因为 RNA打断操作相对简单、快捷， 并且打断的随机性和均一性良好。  With the rapid development of a new generation of high-throughput DNA sequencing technology, RNA sequencing (RNA-seq) has become an important new tool for gene expression and transcriptome analysis. A transcriptome is the sum of all RNAs that a particular cell can transcribe under a certain functional state, including mRNA and non-coding RNA. The current main processes of RNA-Seq include, first, the use of Poly(T) oligonucleotides to extract all RNAs with Poly(A) tails from total RNA, mainly encoding the mRNA transcribed from the gene, and then the resulting The RNA is randomly broken into fragments, the RNA fragments are recovered by ethanol precipitation, and the cDNA fragments are synthesized from the RNA fragments by random primers and reverse transcriptase. Then, the cDNA fragments are end-repaired and ligated to the sequencing adaptor, which will be used for sequencing. cDNA. In this process, RNA was selected to be interrupted first, rather than breaking cDNA after cDNA synthesis because the RNA disruption was relatively simple and fast, and the randomness and uniformity of the break were good.
目前， 常用 RNA断裂试剂将 RNA片段化。 然而， 釆用现阶段的 RNA断裂试剂打 断 RNA时， 步骤繁瑣、 操作复杂、 实验效率低， 不适合批量样品的建库操作， 不利于 整个方法流程的自动化。  Currently, RNA fragmentation reagents are commonly used to fragment RNA. However, when RNA is interrupted by the current RNA fragmentation reagent, the steps are cumbersome, complicated, and the experiment efficiency is low, which is not suitable for the operation of batch samples, which is not conducive to the automation of the entire method.
因此， 目前的 RNA断裂试剂仍有待改进。 发明内容  Therefore, current RNA fragmentation reagents still need to be improved. Summary of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。 为此， 本发明提供了 RNA 断裂试剂及其用途， 以便克服市售的 RNA断裂试剂的缺陷。  The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides RNA fragmentation reagents and uses thereof to overcome the deficiencies of commercially available RNA fragmentation reagents.
根据本发明的一个方面， 本发明提供了一种 RNA断裂试剂。 根据本发明的实施例， 该 According to one aspect of the invention, the invention provides an RNA fragmentation reagent. According to an embodiment of the invention, the
RNA断裂试剂包括： 生物緩冲液， 该生物緩冲液适于将 RNA断裂试剂的 pH值维持在 7~9 之间； 一价金属离子和二价金属离子， 其中， 生物緩冲液、 一价金属离子和二价金属离子 均处于有效浓度。 The RNA fragmentation reagent comprises: a biological buffer, wherein the biological buffer is suitable for maintaining the pH of the RNA fragmentation reagent between 7 and 9; a monovalent metal ion and a divalent metal ion, wherein the biological buffer, one Both the valence metal ion and the divalent metal ion are at an effective concentration.
根据本发明的实施例，在本发明的 RNA断裂试剂中，生物緩冲液的类型不受特别限制。 在本发明中所使用的术语 "生物緩冲液" 是指这样一种液体， 其能够稳定反应体系的 pH, 即其能够及时调节所参与生物反应的反应体系中的 pH, 使该 pH的值维持在反应所需的特 定范围内， 使反应能够顺利进行。 根据本发明的具体示例， 生物緩冲液可以为 Tris緩冲液。 根据本发明的实施例， Tris緩冲液将 RNA断裂试剂的 pH维持在 7至 9之间。 根据本发明 的实施例， Tris緩冲液的浓度不受特别限制， 只要能够实现 Tris緩冲液的作用， 即能够稳定 反应体系的 pH,使本发明的 RNA断裂试剂的 pH维持在 7至 9之间即可。根据本发明的一 些实施例， Tris緩冲液的浓度可以为 100-350mM。根据本发明的一些具体示例， Tris緩冲液 的浓度可以为 150-300mM。 根据本发明的一些实施例， Tris 緩冲液的浓度可以为 200-250mM。 According to an embodiment of the present invention, in the RNA fragmentation reagent of the present invention, the type of the biological buffer is not particularly limited. The term "biological buffer" as used in the present invention refers to a liquid which is capable of stabilizing the pH of a reaction system, that is, it is capable of timely adjusting the pH in a reaction system in which a biological reaction is involved, and the value of the pH is made. Maintain the special required for the reaction Within a certain range, the reaction can proceed smoothly. According to a specific example of the invention, the biological buffer can be a Tris buffer. According to an embodiment of the invention, the Tris buffer maintains the pH of the RNA fragmentation reagent between 7 and 9. According to an embodiment of the present invention, the concentration of the Tris buffer is not particularly limited as long as the action of the Tris buffer can be achieved, that is, the pH of the reaction system can be stabilized, and the pH of the RNA fragmentation reagent of the present invention is maintained at 7 to 9. Just between. According to some embodiments of the invention, the concentration of the Tris buffer may be from 100 to 350 mM. According to some specific examples of the invention, the concentration of the Tris buffer may be from 150 to 300 mM. According to some embodiments of the invention, the concentration of the Tris buffer may be from 200 to 250 mM.
根据本发明的实施例， 在本发明的 RNA断裂试剂中， 一价金属离子可以为 K+和 Na+ 的至少一种。 根据本发明的具体示例， 一价金属离子为 K+。 根据本发明的实施例， 一价金 属离子的浓度可以为 80-400mM。 根据本发明的具体示例， 一价金属离子的浓度可以为 90-300mM。 根据本发明的一些实施例， 一价金属离子的浓度可以为 100-250mM。 根据本发 明的实施例， 二价金属离子可以为 Mg2+和 Zn2+的至少一种。 根据本发明的具体示例， 二价 金属离子为 Mg2+和 Zn2+。 根据本发明的实施例， Mg2+的浓度可以为 8-100mM。 根据本发明 的具体示例， Mg2+的浓度可以为 10-80mM。 根据本发明的一些实施例， Mg2+的浓度可以为 15-60mM。 根据本发明的实施例， Zn2+的浓度可以为 8-100mM。 根据本发明的具体示例， Zn2+的浓度可以为 10-80mM。 根据本发明的一些实施例， Zn2+的浓度可以为 15-60 mM。 According to an embodiment of the present invention, in the RNA fragmentation reagent of the present invention, the monovalent metal ion may be at least one of K+ and Na + . According to a specific example of the invention, the monovalent metal ion is K + . According to an embodiment of the present invention, the concentration of the monovalent metal ion may be 80 to 400 mM. According to a specific example of the present invention, the concentration of the monovalent metal ion may be from 90 to 300 mM. According to some embodiments of the invention, the concentration of monovalent metal ions may range from 100 to 250 mM. According to an embodiment of the present invention, the divalent metal ion may be at least one of Mg 2+ and Zn 2+ . According to a specific example of the invention, the divalent metal ions are Mg 2+ and Zn 2+ . According to an embodiment of the present invention, the concentration of Mg 2+ may be 8-100 mM. According to a specific example of the present invention, the concentration of Mg 2+ may be 10-80 mM. According to some embodiments of the invention, the concentration of Mg 2+ may range from 15 to 60 mM. According to an embodiment of the present invention, the concentration of Zn 2+ may be 8-100 mM. According to a specific example of the present invention, the concentration of Zn 2+ may be 10-80 mM. According to some embodiments of the invention, the concentration of Zn 2+ may range from 15 to 60 mM.
根据本发明的实施例， RNA的来源不受特别限制。 根据本发明的具体示例， RNA可 以来源于原核或真核生物。在本文中所使用的表达方式 "RNA可以来源于原核或真核生物" 是指， 根据本发明实施例的 RNA断裂试剂适于对来源于原核生物和真核生物的 RNA进行 断裂， 从而断裂后的 RNA能够有效地应用于高通量 RNA测序文库的构建。  According to an embodiment of the present invention, the source of RNA is not particularly limited. According to a specific example of the invention, the RNA may be derived from a prokaryotic or eukaryotic organism. The expression "RNA may be derived from a prokaryotic or eukaryotic organism" as used herein means that the RNA fragmentation reagent according to an embodiment of the present invention is suitable for cleavage of RNA derived from prokaryotes and eukaryotes, thereby RNA can be effectively applied to the construction of high-throughput RNA sequencing libraries.
在本文中所使用的术语 "有效浓度" 的含义是指， 生物緩冲液、 金属离子包括一价金 属离子和二价金属离子的浓度能够实现其在相关处理中的相应的功能， 例如， 生物緩冲液 的浓度足以实现将试剂的 pH维持在期望的范围内例如 7-9 , —价金属离子的浓度能够达到 cDNA第一链合成所要的浓度,二价金属离子的浓度能够达到实现打断 RNA所需要的浓度。 根据本发明的实施例， 利用该 RNA断裂试剂进行 RNA断裂， 打断效果与 ABI公司提供的 RNA断裂试剂 （RNA Fragmentation Reagents, AM8740 )相当， 但通过调整打断温度和打 断时间， 利用该试剂能够将 RNA打断为不同长度的片段， 并且利用本发明的 RNA断裂试 剂进行 RNA断裂， 步骤简单、 操作方便、 需时少、 成本低， 且可重复性好。 此外， 发明人 惊奇地发现，才艮据本发明实施例的 RNA断裂试剂能够有效地应用于高通量测序平台的 RNA 测序文库构建， 并适于大批量样本的 RNA测序文库构建。  The term "effective concentration" as used herein means that the concentration of a biological buffer, a metal ion including a monovalent metal ion and a divalent metal ion enables its corresponding function in a related process, for example, a biological The concentration of the buffer is sufficient to maintain the pH of the reagent within a desired range, for example, 7-9, the concentration of the valence metal ion can reach the concentration required for the synthesis of the first strand of the cDNA, and the concentration of the divalent metal ion can achieve the interruption. The concentration required for RNA. According to an embodiment of the present invention, the RNA fragmentation reagent is used for RNA fragmentation, and the breaking effect is equivalent to the RNA Fragmentation Reagents (AM8740) provided by ABI, but the reagent is utilized by adjusting the breaking temperature and the breaking time. The RNA can be interrupted into fragments of different lengths, and the RNA fragmentation reagent of the present invention is used for RNA fragmentation, and the steps are simple, convenient, less time-consuming, low in cost, and reproducible. Furthermore, the inventors have surprisingly found that RNA fragmentation reagents according to embodiments of the present invention can be effectively applied to RNA sequencing library construction of high throughput sequencing platforms and are suitable for RNA sequencing library construction of large samples.
具体地， 根据本发明的实施例， 本发明的 RNA断裂试剂可以包括： 维持 pH值在 7至 9之间的生物緩冲液， 一价金属离子和二价金属离子； 其中生物緩冲液、 一价金属离子和二 价金属离子均处于有效浓度。 其中， 一价金属离子为 cDNA第一链合成所必需， 二价金属 离子为打断 RNA所必需。  Specifically, according to an embodiment of the present invention, the RNA fragmentation reagent of the present invention may comprise: a biological buffer that maintains a pH between 7 and 9, a monovalent metal ion and a divalent metal ion; wherein the biological buffer, Both the monovalent metal ion and the divalent metal ion are at an effective concentration. Among them, monovalent metal ions are necessary for the synthesis of the first strand of cDNA, and divalent metal ions are necessary for interrupting RNA.
根据本发明的实施例， 生物緩冲液可以为 Tris緩冲液， 其浓度可以为 100-350mM, 优 选 150-300mM, 更优选 200-250mM; 将 RNA断裂试剂的 pH维持在 7至 9之间。 根据本发 明的实施例， 一价金属离子可以为 K+和/或 Na+ , 优选 K+; —价金属离子浓度可以为 80-400mM, 优选地 90-300mM, 更优选 100-250mM。 才艮据本发明的实施例， 二价金属阳离 子可以为 Mg2+和 /或 Zn2+, 优选 Mg2+和 Zn2+; Mg2+离子的浓度可以为 8-100mM, 优选 10-80mM, 更优选 15-60mM; Zn2+离子的浓度可以为 8-100mM, 优选 10-80mM, 更优选 15-60mM。 According to an embodiment of the present invention, the biological buffer may be a Tris buffer at a concentration of 100-350 mM, preferably 150-300 mM, more preferably 200-250 mM; maintaining the pH of the RNA fragmentation reagent between 7 and 9 . According to this issue In the embodiment, the monovalent metal ion may be K+ and/or Na + , preferably K+; the valence metal ion concentration may be 80-400 mM, preferably 90-300 mM, more preferably 100-250 mM. According to an embodiment of the present invention, the divalent metal cation may be Mg 2+ and/or Zn 2+ , preferably Mg 2+ and Zn 2+ ; the concentration of Mg 2+ ions may be 8-100 mM, preferably 10-80 mM. More preferably, it is 15-60 mM; the concentration of Zn 2+ ions may be 8-100 mM, preferably 10-80 mM, more preferably 15-60 mM.
此外， 根据本发明的实施例， 在本发明的 RNA断裂试剂中， RNA可以来源于原核或 真核生物， 且可以来源于单个细胞或多个细胞， 单种细胞或多种细胞， 单个组织或多个组 织， 单种组织或多种组织， 一个生物或多个生物， 一种生物或多种生物。  Further, according to an embodiment of the present invention, in the RNA fragmentation reagent of the present invention, RNA may be derived from prokaryotic or eukaryotic organisms, and may be derived from a single cell or a plurality of cells, a single cell or a plurality of cells, a single tissue or Multiple tissues, single tissue or multiple tissues, one organism or multiple organisms, one organism or multiple organisms.
需要说明的是， 根据本发明实施例的 RNA断裂试剂和 ABI公司提供的 RNA断裂试剂 ( RNA Fragmentation Reagents, AM8740 )的打断效果相当， 并且通过调整打断温度和打断 时间， 能够满足对不同片段大小的需求。 将该 RNA断裂试剂应用于 RNA测序文库构建， 其表现出的另外一个优势在于： 断裂 RNA后， 不需要加反应终止剂， 也不需要任何纯化步 骤便能直接进行后续的逆转录反应， 即在特定的逆转录反应体系中， 该断裂试剂能替代第 一链合成緩冲液， 不必另外加入第一链合成緩冲液， 且获得的数据与根据 Illumina mRNA-Seq 文库制备标准流程 （可参见 mRNA Sequencing Sample Preparation Guide , part#l 004898, 通过参照将其全文并入本文）所得到的数据一致， 这表明使用该 RNA断裂 试剂构建 RNA测序文库的方法简化了操作步骤， 能够节约时间， 降低成本， 且在不影响结 果的基础上，为自动化操作创造了非常有利的条件，使自动化批量 RNA样品建库成为可能。  It should be noted that the RNA fragmentation reagent according to the embodiment of the present invention and the RNA Fragmentation Reagents (AM8740) provided by ABI Company have the same breaking effect, and can be satisfied by adjusting the breaking temperature and the breaking time. The size of the fragment needs. The RNA cleavage reagent is applied to the construction of an RNA sequencing library, and the other advantage is that: after cleavage of the RNA, no need to add a reaction terminator, and without any purification step, the subsequent reverse transcription reaction can be directly performed, that is, In a specific reverse transcription reaction system, the cleavage reagent can replace the first strand synthesis buffer without additional addition of the first strand synthesis buffer, and the obtained data is based on the standard procedure for preparation of the Illumina mRNA-Seq library (see mRNA The data obtained by Sequencing Sample Preparation Guide, part #l 004898, which is incorporated herein by reference in its entirety, is consistent, which indicates that the method of constructing an RNA sequencing library using the RNA fragmentation reagent simplifies the operation steps, saving time and reducing costs. And on the basis of not affecting the results, it creates very favorable conditions for automated operation, making it possible to build automated batch RNA samples.
根据本发明的又一方面，本发明提供了根据本发明实施例的 RNA断裂试剂在打断 RNA 中的应用。  According to still another aspect of the present invention, the present invention provides the use of an RNA fragmentation reagent for interrupting RNA according to an embodiment of the present invention.
根据本发明的另一方面， 本发明提供了一种打断 RNA的方法。 根据本发明的实施例， 该方法包括： 使用根据本发明实施例的 RNA断裂试剂打断 RNA。 利用根据本发明实施例 的打断 RNA的方法， 能够方便、 快速、 有效地对原核生物和真核生物的 RNA进行打断， 且成本低、 可重复性好， 并且打断后获得的 RNA片段能够有效地应用于 RNA测序文库的 构建。  According to another aspect of the invention, the invention provides a method of disrupting RNA. According to an embodiment of the invention, the method comprises: disrupting RNA using an RNA fragmentation reagent according to an embodiment of the invention. The method for interrupting RNA according to an embodiment of the present invention can easily, quickly and efficiently interrupt RNA of prokaryotic and eukaryotic organisms, and has low cost, good reproducibility, and RNA fragments obtained after interruption. Can be effectively applied to the construction of RNA sequencing libraries.
根据本发明的再一方面， 本发明提供了一种构建 RNA测序文库的方法。 根据本发明的 实施例， 该方法可以包括以下步骤：  According to still another aspect of the present invention, the present invention provides a method of constructing an RNA sequencing library. According to an embodiment of the invention, the method may comprise the following steps:
( a )使用根据本发明实施例的 RNA断裂试剂打断 RNA, 以便获得 RNA片段。 根据 本发明的实施例， 在 70-100 °C的温度下， 使用 RNA断裂试剂打断 RNA。 根据本发明的具 体示例， 在 85-95°C的温度下， 使用所述 RNA断裂试剂打断 RNA。 根据本发明的一个实施 例， 在 94 °C的温度下， 使用所述 RNA断裂试剂打断 RNA。 根据本发明的实施例， 使用所 述 RNA断裂试剂处理 RNA的作用时间是 3-12min。根据本发明的具体示例，使用所述 RNA 断裂试剂处理 RNA的作用时间是 6-10min。根据本发明的一个实施例，使用所述 RNA断裂 试剂处理 RNA的作用时间是 10min。 根据本发明的实施例， RNA片段的长度为 60-1500nt。 根据本发明的具体示例， RNA 片段的长度为 150-700nt。 根据本发明的一些实施例， RNA 片段的长度为 150-500nt。 ( b )在步骤（ a )后的反应体系中直接加入逆转录酶和随机引物， 进行逆转录， 以便合 成 cDNA第一链。 (a) RNA is disrupted using an RNA cleavage reagent according to an embodiment of the invention to obtain an RNA fragment. According to an embodiment of the invention, RNA is interrupted using an RNA cleavage reagent at a temperature of 70-100 °C. According to a specific example of the invention, the RNA is interrupted using the RNA fragmentation reagent at a temperature of 85-95 °C. According to one embodiment of the invention, the RNA is interrupted using the RNA fragmentation reagent at a temperature of 94 °C. According to an embodiment of the invention, the duration of action of the RNA treatment using the RNA fragmentation reagent is 3-12 min. According to a specific example of the invention, the duration of action of the RNA treatment using the RNA fragmentation reagent is 6-10 min. According to one embodiment of the invention, the duration of action of the RNA treatment using the RNA fragmentation reagent is 10 min. According to an embodiment of the invention, the length of the RNA fragment is between 60 and 1500 nt. According to a specific example of the invention, the length of the RNA fragment is between 150 and 700 nt. According to some embodiments of the invention, the RNA fragments are 150-500 nt in length. (b) Reverse transcriptase and random primers are directly added to the reaction system after the step (a) to perform reverse transcription to synthesize the first strand of cDNA.
( c )在步骤（ b )后的反应体系中加入聚合酶和 cDNA二链合成緩冲液，以便合成 cDNA 第二链并获得双链 cDNA。  (c) adding a polymerase and a cDNA double-strand synthesis buffer to the reaction system after the step (b) to synthesize the second strand of the cDNA and obtain a double-stranded cDNA.
( d ) 纯化双链 cDNA。  (d) Purification of the double-stranded cDNA.
( e )对双链 cDNA进行末端修复、 末端添加碱基 "A" 和连接测序接头， 以便得到连 接产物。 根据本发明的实施例， 可以利用 Klenow片段、 T4 DNA聚合酶和 T4多聚核苷酸 激酶对双链 cDNA进行末端修复， 其中， 该 Klenow片段具有 5，→3，聚合酶活性和 3，→5，聚 合酶活性， 但缺少 5，→3，外切酶活性， 由此， 能够获得经过末端修复的双链 cDNA, 接着可 以利用 Klenow (3'-5' exo-), 即具有 3，→5，外切酶活性的 Klenow, 将经过末端修复的双链 cDNA的 3，末端添加碱基 A,以便获得具有粘性末端 A的双链 cDNA,然后可以利用 T4 DNA 连接酶将具有粘性末端 A的双链 cDNA与测序接头相连， 由此， 能够有效地获得连接产物。  (e) End-repairing the double-stranded cDNA, adding the base "A" to the end, and ligating the sequencing link to obtain the ligated product. According to an embodiment of the present invention, double-stranded cDNA can be end-repaired using Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase, wherein the Klenow fragment has 5, →3, polymerase activity and 3,→ 5, polymerase activity, but lacks 5, → 3, exonuclease activity, thereby obtaining a double-stranded cDNA that has been repaired at the end, and then Klenow (3'-5' exo-), which has 3, → 5. Klenow, which is an exonuclease activity, adds a base A to the 3' end of the double-stranded cDNA which is end-repaired to obtain a double-stranded cDNA having a sticky terminal A, which can then be viscous with a sticky end A using T4 DNA ligase. The double-stranded cDNA is ligated to the sequencing link, whereby the ligation product can be efficiently obtained.
( f )对连接产物进行 PCR扩增， 以便获得扩增产物， 该扩增产物构成 RNA测序文库。 发明人惊奇地发现， 利用根据本发明实施例的构建 RNA测序文库的方法， 能够有效地 构建样本的 RNA测序文库， 且该方法步骤简单、操作方便、 需时少、 成本低、 可重复性好， 并且所得文库质量非常好， 能够有效地应用于高通量测序平台例如 Illumina测序平台，进而 能够有效地对样本进行 RNA测序， 从而基于测序结果， 能够准确有效地确定样本的 RNA 序列信息， 进一步其能够有效地应用于对该样本的基因表达以及转录组的研究。 此外， 根 据本发明的实施例， 本发明的构建 RNA测序文库的方法， 由于步骤简单、 易于操作、 成本 低， 非常适于大批量样本的 RNA测序文库构建， 能够用于大规模、 自动化、 工业化生产。  (f) PCR amplification of the ligation product to obtain an amplification product which constitutes an RNA sequencing library. The inventors have surprisingly found that the method of constructing an RNA sequencing library according to an embodiment of the present invention can efficiently construct an RNA sequencing library of a sample, and the method has the advantages of simple steps, convenient operation, less time, low cost and good repeatability. And the quality of the obtained library is very good, and can be effectively applied to a high-throughput sequencing platform such as the Illumina sequencing platform, thereby enabling efficient RNA sequencing of the sample, thereby accurately and efficiently determining the RNA sequence information of the sample based on the sequencing result, and further It can be effectively applied to the study of gene expression and transcriptome of the sample. In addition, according to an embodiment of the present invention, the method for constructing an RNA sequencing library of the present invention can be used for large-scale, automated, industrialization because of simple steps, easy operation, and low cost, and is very suitable for RNA sequencing library construction of large-scale samples. produce.
具体地， 才艮据本发明的具体示例， 本发明的构建 RNA测序文库的方法， 还可以包括以 下步骤：  Specifically, according to a specific example of the present invention, the method for constructing an RNA sequencing library of the present invention may further comprise the following steps:
( a )使用根据本发明实施例的 RNA断裂试剂断裂 RNA得到 RNA片段；  (a) using an RNA cleavage reagent according to an embodiment of the present invention to cleave RNA to obtain an RNA fragment;
( b )在步骤（ a )后的反应体系中直接加入逆转录酶和随机引物，进行逆转录合成 cDNA 第一链；  (b) directly adding a reverse transcriptase and a random primer to the reaction system after the step (a), and performing reverse transcription to synthesize the first strand of the cDNA;
( c )在步骤（b )后的反应体系中加入聚合酶和 cDNA二链合成緩冲液， 合成 cDNA 第二链， 以便获得双链 cDNA;  (c) adding a polymerase and a cDNA double-strand synthesis buffer to the reaction system after the step (b) to synthesize a second strand of the cDNA to obtain a double-stranded cDNA;
( d ) 纯化步骤（c )所得的双链 cDNA;  (d) purifying the double-stranded cDNA obtained in step (c);
( e )对双链 cDNA进行末端修复， 末端添加碱基 "A" 和连接测序接头， 以便得到连 接产物；  (e) end-repairing the double-stranded cDNA, adding a base "A" at the end and ligating the sequencing link to obtain a ligation product;
( f )将步骤（ e )所得的连接产物进行 PCR扩增， 以便获得 RNA测序文库。  (f) PCR-amplifying the ligation product obtained in step (e) to obtain an RNA sequencing library.
根据本发明的实施例，其中利用 RNA断裂试剂打断 RNA的作用温度可以是 70- 100 °C , 优选 85-95 °C , 更优选 94°C。 根据本发明的一些实施例， 利用 RNA断裂试剂打断 RNA的 作用时间可以是 3-12min, 优选 6-10min, 更优选 10min。  According to an embodiment of the present invention, the action temperature at which RNA is interrupted by the RNA fragmentation reagent may be 70 to 100 ° C, preferably 85 to 95 ° C, more preferably 94 ° C. According to some embodiments of the invention, the duration of action of RNA disruption using an RNA cleavage reagent can be from 3 to 12 min, preferably from 6 to 10 min, more preferably 10 min.
根据本发明的一些实施例， 其中步骤（a )中所得的 RNA片段的长度可以为 60-1500nt, 优选 150-700nt, 更优选 150-500nt。 根据本发明的又一方面， 本发明提供了一种 RNA测序文库。 根据本发明的实施例， 该 RNA测序文库是由本发明的构建 RNA测序文库的方法构建的。 发明人发现， 根据本发明 实施例的 RNA测序文库， 能够有效地应用于高通量测序平台例如 Illumina测序平台， 进而 基于测序结果， 能够准确有效地确定样本的 RNA序列信息， 从而基于所得的信息， 能够有 效地对该样本进行基因表达以及转录组研究。 According to some embodiments of the invention, wherein the RNA fragment obtained in step (a) may have a length of from 60 to 1500 nt, preferably from 150 to 700 nt, more preferably from 150 to 500 nt. According to yet another aspect of the invention, the invention provides an RNA sequencing library. According to an embodiment of the invention, the RNA sequencing library is constructed by the method of the invention for constructing an RNA sequencing library. The inventors have found that an RNA sequencing library according to an embodiment of the present invention can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and based on the sequencing result, can accurately and efficiently determine the RNA sequence information of the sample, thereby based on the obtained information. , can effectively carry out gene expression and transcriptome research on the sample.
根据本发明的另一方面， 本发明提供了一种测序方法。 根据本发明的实施例， 该方法 可以包括以下步骤： 才艮据本发明实施例的构建 RNA测序文库的方法构建 RNA测序文库； 以及对 RNA测序文库进行测序， 其中， 测序使用高通量测序平台进行， 该高通量测序平台 为选自 Illumina/Solexa、 ABI Solid和 Roche 454测序平台的至少一种。  According to another aspect of the invention, the invention provides a sequencing method. According to an embodiment of the present invention, the method may comprise the steps of: constructing an RNA sequencing library according to the method of constructing an RNA sequencing library according to an embodiment of the present invention; and sequencing the RNA sequencing library, wherein the sequencing uses a high-throughput sequencing platform Performed, the high throughput sequencing platform is at least one selected from the group consisting of Illumina/Solexa, ABI Solid, and Roche 454 sequencing platforms.
根据本发明的实施例， 利用本发明的测序方法能够方便有效地构建样本的 RNA测序文 库， 并能够准确地对该 RNA测序文库进行测序， 并且该方法步骤简单、 易于操作、 需时少、 成本低， 可重复性好、 所得测序结果准确可靠， 由此， 该方法能够应用于大量样本的 RNA 测序， 并且能够推广应用于大规模的工业自动化生产。  According to an embodiment of the present invention, the sequencing method of the present invention can conveniently and efficiently construct an RNA sequencing library of a sample, and can accurately sequence the RNA sequencing library, and the method is simple, easy to operate, less time-consuming, and cost-effective. Low, repeatable, and accurate and reliable sequencing results, the method can be applied to RNA sequencing of a large number of samples, and can be applied to large-scale industrial automation production.
具体地， 根据本发明的具体示例， 本发明的测序方法还可以包括构建根据本发明实施 例的 RNA测序文库， 并对该测序文库进行测序的步骤。 才艮据本发明的实施例， 测序可通过 任何测序方法进行， 包括但不限于双脱氧链终止法； 优选高通量的测序方法， 包括但不限 于第二代测序平台或者是单分子测序平台。 其中， 第二代测序平台 （可参见 Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 11(1): 31-46, 2010, 通过参照将其 全文并入本文）包括但不限于 Illumina/Solexa( GATM, HiSeq2000TM等）、 ABI Solid和 Roche 454 (焦磷酸测序）测序平台； 单分子测序平台 （技术） 包括但不限于 Helicos公司的真实 单分子测序技术 ( True Single Molecule DNA sequencing )、 Pacific Biosciences公司的单分子 实时测序 ( single molecule real-time (SMRT™) )以及 Oxford Nanopore Technologies公司的纳 米孑 则序技术等 (可参见 Rusk, Nicole. Cheap Third-Generation Sequencing. Nature Methods. 6(4): 244-244, 2009, 通过参照将其全文并入本文）。  Specifically, according to a specific example of the present invention, the sequencing method of the present invention may further comprise the step of constructing an RNA sequencing library according to an embodiment of the present invention and sequencing the sequencing library. According to an embodiment of the invention, sequencing can be performed by any sequencing method, including but not limited to the dideoxy chain termination method; preferably a high throughput sequencing method, including but not limited to a second generation sequencing platform or a single molecule sequencing platform . Among them, the second generation sequencing platform (see Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 11(1): 31-46, 2010, which is incorporated herein by reference in its entirety) including but not limited to Illumina/ Solexa (GATM, HiSeq2000TM, etc.), ABI Solid and Roche 454 (pyrophosphate sequencing) sequencing platforms; single molecule sequencing platforms (technologies) including but not limited to Helicos' True Single Molecule DNA sequencing, Pacific Biosciences The company's single molecule real-time (SMRTTM) and Oxford Nanopore Technologies' nanometer-order technology (see Rusk, Nicole. Cheap Third-Generation Sequencing. Nature Methods. 6(4): 244-244, 2009, which is incorporated herein by reference in its entirety.
需要说明的是， 本发明的 RNA断裂试剂及其用途， 是本申请的发明人经过艰苦的 创造性劳动劳动和优化的工作才完成的。 本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明  It should be noted that the RNA fragmentation reagent of the present invention and its use are completed by the inventor of the present application through arduous creative labor and optimization work. The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：  The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from
图 1： 显示了对照例中的 Illumina mRNA-Seq文库制备流程示意图；  Figure 1: Schematic diagram showing the preparation process of the Illumina mRNA-Seq library in the comparative example;
图 2: 显示了实施例 1中根据本发明的 mRNA-Seq文库制备流程示意图； 图 3: 显示了对照例中所得的 RNA片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC- Agilent,用本发明对照例中 Illumina测序平台提供的 5xRNA 断裂试剂在 94 °C断裂 5min所得； Figure 2: shows a schematic diagram of the preparation process of the mRNA-Seq library according to the present invention in Example 1; Figure 3: Agilent Bioanalyzer 2100 detection chart showing the size of the RNA fragment obtained in the control example, Wherein the RNA fragment was obtained from MAQC-Agilent, and cleavage was carried out at 94 ° C for 5 min using the 5x RNA cleavage reagent provided by the Illumina sequencing platform of the control of the present invention;
图 4: 显示了实施例 1中所得的 RNA片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC- Agilent ,用本发明实施例 1中的 3xRNA断裂试剂在 94 °C断 裂 3min所得；  Figure 4: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained in Example 1, wherein the RNA fragment was obtained from MAQC-Agilent, and was cleaved at 94 °C for 3 min using the 3xRNA fragmentation reagent of Example 1 of the present invention;
图 5 : 显示了实施例 1中所得的 RNA片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC- Agilent ,用本发明实施例 1中的 3xRNA断裂试剂在 94 °C断 裂 6min所得；  Figure 5: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained in Example 1, wherein the RNA fragment was obtained from MAQC-Agilent, and was cleaved at 94 °C for 6 min using the 3xRNA cleavage reagent of Example 1 of the present invention;
图 6: 显示了实施例 1中所得的 RNA片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC-Agilent ,用本发明实施例 1中的 3xRNA断裂试剂在 94 °C断 裂 12min所得；  Figure 6: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained in Example 1, wherein the RNA fragment was obtained from MAQC-Agilent, and was cleaved at 94 °C for 12 min with the 3x RNA fragmentation reagent of Example 1 of the present invention;
图 7: 显示了实施例 11中利用 3xRNA断裂试剂在 94 °C断裂 lOmin所得的 RNA片 段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC-AB ;  Figure 7: Agilent Bioanalyzer 2100 assay showing the RNA fragment size obtained by cleavage of 10x RNA fragmentation reagent at 94 °C for 10 min in Example 11, wherein the RNA fragment was from MAQC-AB;
图 8: 显示了实施例 11中利用 3xRNA断裂试剂在 94 °C断裂 lOmin所得的 RNA片 段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC-AB平行样； 图 9: 显示了实施例 11中利用 3xRNA断裂试剂在 94 °C断裂 lOmin所得的 RNA片 段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC-Agilent;  Figure 8: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3x RNA fragmentation reagent in Example 11, wherein the RNA fragment is from the MAQC-AB parallel sample; Figure 9: shows Example 11 Agilent Bioanalyzer 2100 assay image of RNA fragment size obtained by cleavage of 10 min at 94 °C using a 3x RNA cleavage reagent, wherein the RNA fragment was from MAQC-Agilent;
图 10: 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC-Agilent平行 样；  Figure 10: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3xRNA cleavage reagent in Example 11, wherein the RNA fragment was from a MAQC-Agilent parallel sample;
图 11 : 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自玉米 RNA;  Figure 11: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3x RNA fragmentation reagent in Example 11, wherein the RNA fragment is derived from maize RNA;
图 12: 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自玉米 RNA平行样； 图 13 : 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自水稻 RNA;  Figure 12: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3xRNA cleavage reagent in Example 11, wherein the RNA fragment is from a parallel sample of maize RNA; Figure 13: shows the use in Example 11 Agilent Bioanalyzer 2100 assay of the size of the RNA fragment obtained by cleavage of the 3xRNA fragmentation reagent at 94 ° C for 10 min, wherein the RNA fragment is derived from rice RNA;
图 14: 显示了实施例 11中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得实施例 1 中所得的 RNA片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自水稻 RNA平行样；  Figure 14: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained in Example 1 using the 3xRNA cleavage reagent at 94 °C for 10 min, in which the RNA fragment was derived from a parallel sample of rice RNA;
图 15 : 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自真菌 RNA;  Figure 15: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3x RNA fragmentation reagent in Example 11, wherein the RNA fragment is derived from fungal RNA;
图 16: 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自真菌 RNA平行样； 图 17 : 显示了实施例 11 中利用 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自小鼠 RNA;  Figure 16: Agilent Bioanalyzer 2100 assay showing the size of the RNA fragment obtained by cleavage of 10 min at 94 °C using a 3x RNA fragmentation reagent in Example 11, wherein the RNA fragment is from a fungal RNA parallel; Figure 17: shows the use in Example 11 The Agilent Bioanalyzer 2100 assay of the RNA fragment size obtained by cleavage of the 3xRNA fragmentation reagent at 94 ° C for 10 min, wherein the RNA fragment is derived from mouse RNA;
图 18: 显示了实施例 11中的 3xRNA断裂试剂在 94°C断裂 lOmin所得的 RNA片 段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自小鼠 RNA平行样。 具体实施方式 Figure 18: shows the RNA fragment obtained by breaking the 3x RNA fragmentation reagent of Example 11 at 94 ° C for 10 min. The segment size of the Agilent Bioanalyzer 2100 assay, in which the RNA fragments were derived from mouse RNA parallel. detailed description
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。  The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative only and not to limit the invention.
此外， 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件 (例如参考 j.萨姆布鲁克等著， 黄培堂等译的《分子克隆实验指南》， 第三版， 科学出版社） 或者按照产品说明书进行。 所用试剂或仪器未注明生产厂商者， 均为可以通过市购获得的 常规产品， 例如可以釆购自 Illumina公司。  In addition, in the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., "Molecular Cloning Experimental Guide", third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer. They are all commercially available products, such as those available from Illumina.
实施例 1:  Example 1:
釆用本发明的构建 RNA测序文库的方法， 构建样品的 RNA测序文库。 其中， 为确定 较优的 RNA打断条件， 釆用同一 RNA断裂试剂对相同的样本进行试验， 设置 5个断裂温 度梯度为 70°C、 80°C、 90°C、 94°C和 100 °C , 6个断裂时间梯度为 1.5min、 3min、 6min、 8min、 lOmin和 12min, 共 30种方案。 具体实险步骤如下所述：  Using the method of constructing an RNA sequencing library of the present invention, an RNA sequencing library of a sample is constructed. In order to determine the optimal RNA breaking conditions, the same sample was tested with the same RNA fragmentation reagent, and five fracture temperature gradients of 70 ° C, 80 ° C, 90 ° C, 94 ° C, and 100 ° were set. C, 6 rupture time gradients were 1.5 min, 3 min, 6 min, 8 min, lOmin and 12 min, a total of 30 programs. The specific risk steps are as follows:
1.1样品试剂  1.1 sample reagent
MAQC-Agilent( Universal Human Reference RNA, UHRR, Stratagen); MAQC-AB( Human Brain Reference RNA, Ambion); 3xRNA断裂试剂： 200mM Tris-HCl ( H 8.0 ), lOOmM KC1, 15mM MgCl2, 15mM ZnCl2 (断裂试剂的各组分均购自 Sigma, 试剂不含 RNA酶）； 其它各 试剂緩冲液， 除另有标明， 均来自 Illumina公司提供的 mRNA-Seq样品制备试剂盒。 MAQC-Agilent (Universal Human Reference RNA, UHRR, Stratagen); MAQC-AB (Human Brain Reference RNA, Ambion); 3x RNA fragmentation reagent: 200 mM Tris-HCl (H 8.0 ), 100 mM KC1, 15 mM MgCl 2 , 15 mM ZnCl 2 ( Each component of the fragmentation reagent was purchased from Sigma, and the reagent contained no RNase); each reagent buffer, unless otherwise indicated, was obtained from the mRNA-Seq sample preparation kit supplied by Illumina.
1.2实险步骤  1.2 actual steps
1.2.1纯化 mRNA  1.2.1 Purification of mRNA
1 )取 l-l(Vg总 RNA( MAQC- Agilent或 MAQC-AB )至一个无 RNA酶的 EP管（ Axygen ) 中， 用 DEPC水（ Ambion )稀释至 50μ1, 混匀后于 65°C下变性 5分钟以打开二级结构， 然 后立即将样品置于水上。  1) Take ll (Vg total RNA (MAQC-Agilent or MAQC-AB) into an RNase-free EP tube (Axygen), dilute to 50μ1 with DEPC water (Amion), mix and denature at 65 °C. Minutes to open the secondary structure, then immediately place the sample on the water.
2 )吸取 15μ1 Sera-Mag oligo(dT)磁珠（ Invitrogen )于 1.5ml的不粘 EP管（ non-stick-EP ) 中， 用 ΙΟΟμΙ结合緩冲液洗涤磁珠两次， 然后将磁珠重悬于 50μ1结合緩冲液中， 再将第一 步中制得的总 RNA加入管中， 于室温下放置 5min。  2) Pipette 15μ1 Sera-Mag oligo(dT) magnetic beads (Invitrogen) in 1.5ml non-stick-EP tube, wash the magnetic beads twice with ΙΟΟμΙ binding buffer, then weigh the beads The cells were suspended in 50 μl of binding buffer, and the total RNA prepared in the first step was added to the tube and allowed to stand at room temperature for 5 minutes.
3 )将不粘 EP管置于磁分离器（MPC, Invitrogen )上 2min, 去除上清， 再用 200μ1洗 脱緩冲液清洗磁珠两次。 然后取一新的不粘的 ΕΡ管， 加入 50μ1的结合緩冲液， 备用。  3) The non-stick EP tube was placed on a magnetic separator (MPC, Invitrogen) for 2 min, the supernatant was removed, and the magnetic beads were washed twice with 200 μl elution buffer. Then take a new non-stick tube and add 50μ1 of binding buffer for later use.
4 ) 向含磁珠的 ΕΡ管中加入 50μ1 10mM Tris-HCl, 然后于 80°C下加热 2min将 mRNA 从磁珠上洗脱下来， 再迅速将 EP管转至磁分离器上。 然后将 mRNA转移至上步中备用的 加有 50μ1结合緩冲液的 ΕΡ管， 混合后于 65 °C变性 5min , 打开二级结构， 然后立即将样品 置于水上。 另外， 立即将 200μ1洗脱緩冲液加入到含有磁珠的 EP管中， 洗两次磁珠。  4) Add 50 μl of 10 mM Tris-HCl to the magnetic tube-containing helium tube, then elute the mRNA from the magnetic beads by heating at 80 ° C for 2 min, and then quickly transfer the EP tube to the magnetic separator. The mRNA was then transferred to a spare tube supplemented with 50 μl of binding buffer in the previous step, mixed and denatured at 65 °C for 5 min, the secondary structure was opened, and the sample was immediately placed on water. In addition, 200 μl of the elution buffer was immediately added to the EP tube containing the magnetic beads, and the magnetic beads were washed twice.
5 )将 ΙΟΟμΙ mRNA样品加入含有洗过两次的磁珠的 EP管中， 室温放置 5min, 将 EP 管置于 MPC上 2min, 小心吸除上清， 再用 200μ1洗脱緩冲液清洗磁珠两次。 6 )向含磁珠的 EP管中加入 17μ1 10mM Tris-HCl, 80°C加热 2min, 从而将 mRNA从磁 珠上洗脱下来。迅速地将 EP管转至 MPC上，转移 mRNA洗脱液至一新的 200μ1 PCR管中， 回到收大约 16μ1 ητΚ_ΝΑ。 5) Add the ΙΟΟμΙ mRNA sample to the EP tube containing the magnetic beads washed twice, leave it at room temperature for 5 min, place the EP tube on the MPC for 2 min, carefully aspirate the supernatant, and then wash the beads with 200 μl elution buffer. twice. 6) 17 μl of 10 mM Tris-HCl was added to the EP tube containing the magnetic beads, and heated at 80 ° C for 2 min to elute the mRNA from the magnetic beads. The EP tube was quickly transferred to the MPC, and the mRNA eluate was transferred to a new 200 μl PCR tube and returned to approximately 16 μl ητΚ_ΝΑ.
1.2.2断裂 mRNA及合成 cDNA第一链  1.2.2 cleavage mRNA and synthesis of the first strand of cDNA
往回收到的 16μ1 mRNA溶液中添加 5.1μ1 3xRNA 断裂试剂， 94 °C lOmin (或 70°C 1.5min或 70°C 3min或 70°C 6min或 70°C 8min或 70°C lOmin或 70°C 12min,其它温度 80 °C、 90°C、 94°C和 100 °C设置作用时间同 70°C )进行断裂反应， 然后立即置于水上，添加 Ιμΐ 随机引物， 并于 65 °C下反应 5min以打开单链二级结构， 然后置于水上。  Add 5.1μ1 3xRNA cleavage reagent to the recovered 16μ1 mRNA solution, 94°C lOmin (or 70°C 1.5min or 70°C 3min or 70°C 6min or 70°C 8min or 70°C lOmin or 70°C 12min, other temperature 80 °C, 90 °C, 94 °C and 100 °C set the action time with 70 °C) to carry out the fracture reaction, and then immediately placed on the water, add Ιμΐ random primer, and react at 65 °C for 5min To open the single-chain secondary structure and then place it on the water.
配置包括 2μ1 lOOmM DTT、 0.4μ125mM dNTP混合底物、 0.5μ1 RNA抑制剂的反应混合 物， 然后将该混合物加入含 RNA的管中， 混匀后于室温下放置 2min, 然后加入 Ιμΐ 200υ/μ1 Superscript II逆转录酶混匀， 总体系为 25μ1。 然后， 在 PCR仪上按照以下程序进行反应： 步骤 1 25 °C lOmin  The reaction mixture consisting of 2 μl lOOmM DTT, 0.4 μ125 mM dNTP mixed substrate, 0.5 μl RNA inhibitor, and then the mixture was added to an RNA-containing tube, mixed and allowed to stand at room temperature for 2 min, then Ιμΐ 200υ/μ1 Superscript II was added. The reverse transcriptase was mixed and the total system was 25 μl. Then, follow the procedure below on the PCR machine: Step 1 25 °C lOmin
步骤 2 42 °C 50min  Step 2 42 °C 50min
步骤 3 70 °C 15min  Step 3 70 °C 15min
步骤 4 4°C 保持  Step 4 4 °C hold
由此， 合成 cDNA第一链。  Thus, the first strand of cDNA was synthesized.
1.2.3合成 cDNA第二链  1.2.3 Synthesis of the second strand of cDNA
向上一步的反应体系中补水至 82.8μ1, 然后依次加入 ΙΟμΙ ϋΕΧ二链合成緩冲液、 1.2μ1 25mM dNTP混合物， 混匀后于水上放置 5min, 再加入 Ιμΐ RNaseH、 5μ1 DNA聚合酶， 混 匀后将反应管置于 16°C下反应 2.5小时。  To the next step of the reaction system, doubling the water to 82.8μ1, then adding ΙΟμΙ ϋΕΧ two-strand synthesis buffer, 1.2μ1 25mM dNTP mixture, mixing and placing on the water for 5min, then adding Ιμΐ RNaseH, 5μ1 DNA polymerase, and mixing. The reaction tube was placed at 16 ° C for 2.5 hours.
反应完成后， 用 QIAquick PCR纯化试剂盒 ( Qiagen )纯化双链 cDNA产物， 并将其溶 于 50μ1洗脱液， 以便获得 50μ1 DNA溶液。  After completion of the reaction, the double-stranded cDNA product was purified using QIAquick PCR Purification Kit (Qiagen), and dissolved in 50 μl of the eluate to obtain a 50 μl DNA solution.
1.2.4末端修复  1.2.4 end repair
向上述得到 50μ1 ϋΝΑ溶液中依次加入 27.4μ1水、 ΙΟμΙ 10x末端修复緩冲液、 1.6μ125 mM dNTP混合物、 5μ1 Τ4 DNA聚合酶、 Ιμΐ Klenow DNA聚合酶、 5μ1 Τ4 ΡΝΚ, 得到 ΙΟΟμΙ末 端修复的总反应体系， 然后将反应管置于 20 °C下反应 30min。  To the above-obtained 50 μl ϋΝΑ solution, 27.4 μl of water, ΙΟμΙ 10× terminal repair buffer, 1.6 μl of 125 mM dNTP mixture, 5 μl of DNA4 DNA polymerase, Ιμΐ Klenow DNA polymerase, and 5 μl Τ4 依次 were sequentially added to obtain a total reaction of ΙΟΟμΙ end repair. The system was then reacted at 20 ° C for 30 min.
反应完成后， 用 QIAquick PCR纯化试剂盒（Qiagen )纯化末端修复产物， 并将其溶于 32μ1洗脱液， 以便获得 32μ1 DNA溶液。  After completion of the reaction, the terminal repair product was purified by QIAquick PCR Purification Kit (Qiagen) and dissolved in a 32 μl eluate to obtain a 32 μl DNA solution.
1.2.5力口 A, 加接头  1.2.5 force port A, add connector
向上述得到的 32μ1 DNA溶液中依次加入 5μ1 A-Tailing反应液、 ΙΟμΙ 1 mM dATP、 3μ1 To the 32μ1 DNA solution obtained above, 5μ1 A-Tailing reaction solution, ΙΟμΙ 1 mM dATP, 3μ1 were sequentially added.
Klenow exo (3' to 5'外切酶)， 得到 50μ1加 Α的总反应体系， 然后将反应管置于 37 °C下反应 30 min, 以便获得加 A产物。 Klenow exo (3' to 5' exonuclease), a total reaction system of 50 μl plus hydrazine was obtained, and then the reaction tube was placed at 37 ° C for 30 min to obtain an A-added product.
反应完成后， 用 MinElute PCR纯化试剂盒（Qiagen )纯化加 A产物， 并将其溶于 23μ1 洗脱液。  After completion of the reaction, the product of Add A was purified by MinElute PCR Purification Kit (Qiagen) and dissolved in a 23 μl eluate.
向上述得到的 23μ1加 Α产物中依次加入 25μ12xT4 DNA快速连接緩冲液、 Ιμΐ ΡΕ接头 混合物和 1μ1 Τ4 DNA连接酶， 得到 50μ1加接头的总反应体系， 然后将反应管置于室温反 应 15 min, 以便获得连接产物。 To the 23 μl addition product obtained above, 25 μl of Tx DNA rapid ligation buffer, Ιμΐ ΡΕ linker mixture and 1 μl of DNA4 DNA ligase were sequentially added to obtain a total reaction system of 50 μl plus a linker, and then the reaction tube was placed at room temperature. It should be 15 min in order to obtain the ligation product.
反应完成后， 用 MinElute PCR纯化试剂盒（Qiagen ) 纯化连接产物， 并将其溶于 ΙΟμΙ 洗脱液。  After completion of the reaction, the ligation product was purified using a MinElute PCR Purification Kit (Qiagen) and dissolved in a ΙΟμΙ eluate.
1.2.6连接产物胶纯化  1.2.6 purification of the linker product
配制 2 %的琼脂糖凝胶， 选择 lOObp DNA Ladder, 将上述获得的连接产物进行 120v电 泳 60min, 然后切胶回收。 根据接头以及所需目的片段大小决定， 切胶范围为 200-400bp。 然后利用 QIAquick胶回收试剂盒（Qiagen )将切下的胶块进行回收， 最后将回收的连接产 物溶于 30μ1洗脱液。  A 2% agarose gel was prepared, and a lOObp DNA Ladder was selected, and the ligation product obtained above was subjected to 120v electrophoresis for 60 minutes, and then the gel was recovered. The size of the cut is 200-400 bp depending on the size of the linker and the desired size of the fragment. The cut pieces were then recovered using a QIAquick Glue Recovery Kit (Qiagen), and the recovered ligation product was finally dissolved in a 30 μl eluate.
1.2.7 PCR扩增及纯化  1.2.7 PCR amplification and purification
向上述得到的 30μ1回收的连接产物中依次加入 l(V1 5xPhusion緩冲液、 l l PCR Primer To the 30 μl recovered ligation product obtained above, l (V1 5xPhusion buffer, l l PCR Primer)
PE 1.0、 Ιμΐ PCR Primer PE 2.0、 0.5μ1 25 mM dNTP混合物、 0.5μ1 Phusion DNA聚合酶和 7μ1 水， 得到 50μ1 PCR扩增的总反应体系， 然后在 PCR仪上按照以下程序进行反应： PE 1.0, Ιμΐ PCR Primer PE 2.0, 0.5 μl 25 mM dNTP mixture, 0.5 μl Phusion DNA polymerase and 7 μl water were used to obtain a total reaction system of 50 μl PCR amplification, and then reacted on a PCR machine according to the following procedure:
a. 在 98摄氏度下 30秒  a. 30 seconds at 98 degrees Celsius
b. 15个循环的：  b. 15 cycles:
98 °C下 10秒  10 seconds at 98 °C
65摄氏度下 30秒  30 seconds at 65 degrees Celsius
72摄氏度下 30秒  30 seconds at 72 degrees Celsius
c 72摄氏度下 5分钟  c 72 degrees Celsius for 5 minutes
d. 保持在 4摄氏度下  d. Keep at 4 degrees Celsius
由此， 获得扩增产物。  Thereby, an amplification product was obtained.
接着， 利用 QIAquick PCR纯化试剂盒（Qiagen )纯化扩增产物， 并将其溶于 32μ1洗脱 液中， 由此， 该扩增产物构成 RNA测序文库。  Next, the amplified product was purified using QIAquick PCR Purification Kit (Qiagen) and dissolved in a 32 μl eluate, whereby the amplified product constituted an RNA sequencing library.
然后， 利用 Agilent Bioanalyzer 2100对通过各个方案获得的 RNA片段进行检测， 并对 通过 3xRNA断裂试剂 94°C处理 lOmin制得的样品 MAQC-AB和 MAQC-Agilent的 RNA测 序文库模板进行 Illumina Hiseq2000测序， 其中测序数据统计结果见下述表 1。  Then, the RNA fragment obtained by each protocol was detected by Agilent Bioanalyzer 2100, and the Illumina Hiseq2000 template was sampled by the RNA sequencing library templates of samples MAQC-AB and MAQC-Agilent prepared by treating the 10x RNA fragmentation reagent at 94 ° C for 94 min. The statistical results of the sequencing data are shown in Table 1 below.
其中， 图 4-6显示了以 MAQC-Agilent RNA为样本， 3xRNA断裂试剂 94 °C下分别断裂 RNA 3min、 6min及 12min后所得目的片段的 Agilent Bioanalyzer 2100检测图， 由图可知， RNA被打断成相对集中的片段，但从片段大小的覆盖度来看， 图 4显示断裂 3min仍然存在 大量未被打断或者没有完全打断的 RNA, 图 5显示断裂 6min效果有所改善， 但仍有 700bp 左右的 RNA片段， 图 6显示打断 12min, 形成的主峰比较集中， 但是由于打断时间片长， RNA片段相对稍小。  Among them, Figure 4-6 shows the Agilent Bioanalyzer 2100 detection image of the target fragment obtained by cleavage of RNA at 94 °C for 3 min, 6 min and 12 min with MAQC-Agilent RNA as sample. The RNA is interrupted. In a relatively concentrated segment, but from the coverage of the fragment size, Figure 4 shows that there are still a large number of unbroken or not completely interrupted RNA for 3 min. Figure 5 shows that the effect of 6 min is improved, but there is still 700 bp. The left and right RNA fragments, Figure 6 shows that the main peak formed was relatively concentrated after 12 min of interruption, but the RNA fragment was relatively small due to the length of the interrupted time.
此外，结合本实施例其他方案所得的 RNA片段的 Agilent Bioanalyzer 2100检测图可知， 对于样本 MAQC-Agilent或 MAQC-AB , 在各个作用温度下， 断裂 1.5min不能将 RNA打断 成相对较集中的片段， 作用 3min、 6min或 8min时产生的目的片段中存在不同程度的大片 段， 而断裂 12min时目的片段相对较小， 从峰图的覆盖度和文库的信息分析要求来看， 利 用该试剂断裂不同样本 RNA的时间可在 3-12min范围内调整。同样的 MAQC-Agilent样本， 在固定的断裂时间如 lOmin, 断裂温度为 70°C , 80 °C , 90°C或 100 °C时效果不如或近似于 94 °C (图谱未显示）。 因此， 对于样本 MAQC-Agilent或 MAQC-AB, 结合考虑断裂温度和 时间对 RNA 片段的大小范围集中程度的影响， 选择较优的断裂试剂作用条件为 94 °C 10min。 对照例： In addition, the Agilent Bioanalyzer 2100 test chart of the RNA fragment obtained by the other schemes of the present example shows that for the sample MAQC-Agilent or MAQC-AB, the RNA can not be broken into relatively concentrated fragments at the respective action temperatures for 1.5 min. There are different degrees of large fragments in the target fragment produced at 3min, 6min or 8min, and the target fragment is relatively small when the fragmentation is 12min. From the coverage of the peak map and the information analysis requirements of the library, the fragmentation is different with the reagent. The time of the sample RNA can be adjusted within the range of 3-12 min. The same MAQC-Agilent sample, At a fixed rupture time of 10 min, a break temperature of 70 ° C, 80 ° C, 90 ° C or 100 ° C is not as good as or close to 94 ° C (not shown). Therefore, for the sample MAQC-Agilent or MAQC-AB, considering the effect of the breaking temperature and time on the concentration range of the RNA fragment size, the optimal cleavage reagent was selected to be 94 °C for 10 min. Control example:
釆用 Illumina测序平台提供的构建 RNA测序文库的方法， 构建样品的 RNA测序文库。 具体实验材料及实验步骤如下所述：  构建 Construct an RNA sequencing library of the sample using the method of constructing an RNA sequencing library provided by the Illumina sequencing platform. The specific experimental materials and experimental procedures are as follows:
样品试剂：  Sample reagent:
5xRNA断裂緩冲液（ Applied Biosystem ); 其他材料、 试剂或緩冲液的来源同实施例 1 中的步骤 1.1。  5xRNA cleavage buffer (Applied Biosystem); the source of other materials, reagents or buffers is the same as step 1.1 in Example 1.
操作步骤：  Steps:
1 ) 纯化 mRNA, 操作步骤同实施例 1中的步骤 1.2.1；  1) Purification of mRNA, the procedure is the same as step 1.2.1 in Example 1;
2 ) 断裂 mRNA  2) rupture mRNA
向上述步骤的反应体系中加入 4μ1 5xRNA断裂緩冲液， 于 94°C反应 5min, 然后立即加 入 2μ1终止反应液， 随后加入 3Μ NaAC ( ρΗ 5.2 )、 糖元、 100%乙醇， 混匀后置于 -80°C下 30min, 沉淀出 RNA。  Add 4 μl 5×RNA cleavage buffer to the reaction system of the above step, react at 94 ° C for 5 min, then immediately add 2 μl to terminate the reaction solution, then add 3 Μ NaAC ( ρΗ 5.2 ), glycogen, 100% ethanol, and mix. RNA was precipitated at -80 ° C for 30 min.
3 )合成 cDNA第一链  3) Synthesis of the first strand of cDNA
将 RNA沉淀溶于 DEPC水中进行第一链合成反应。第一链合成体系如下： 11.1 μΙ ΚΝΑ, 随机引物 Ιμΐ, 65 °C 5min打开单链二级结构， 置于水上。 配置反应混合物， 包括 4μ1 5X第 一链緩冲液、 2μ1 lOOmM DTT^ 0.4μ1 25mM dNTP Mix、 0.5μ1 RNA酶抑制剂将混合物加入 含 RNA的管中， 混匀后室温放置 2min, 然后加入 Ιμΐ Superscript II逆转录酶（ 200U/ μ 1 ) 混匀， 总体系 20μ1。 在 PCR仪上按照以下程序进行反应：  The RNA precipitation was dissolved in DEPC water for the first strand synthesis reaction. The first strand synthesis system was as follows: 11.1 μΙ ΚΝΑ, random primer Ιμΐ, open the single-stranded secondary structure at 65 °C for 5 min, placed on water. Configure the reaction mixture, including 4μ1 5X first strand buffer, 2μl lOOmM DTT^ 0.4μ1 25mM dNTP Mix, 0.5μ1 RNase inhibitor, add the mixture to the RNA-containing tube, mix and let stand for 2min at room temperature, then add Ιμΐ Superscript II reverse transcriptase (200 U / μ 1 ) was mixed, the total system was 20 μl. The reaction was carried out on the PCR machine according to the following procedure:
步骤 1 25 °C lOmin  Step 1 25 °C lOmin
步骤 2 42 °C 50min  Step 2 42 °C 50min
步骤 3 70 °C 15min  Step 3 70 °C 15min
步骤 4 4°C 保持  Step 4 4 °C hold
后续操作步骤同实施例 1中的步骤 1.2.3~1.2.7。  The subsequent operation steps are the same as steps 1.2.3 to 1.2.7 in Embodiment 1.
反应完成后， 用 QIAquick PCR纯化试剂盒 ( Qiagen ) 纯化 PCR产物， 溶于 32μ1洗脱 液， 并用 Agilent Bioanalyzer 2100和 Illumina Hiseq2000对纯化产物进行检测和测序。 其中， 测序结果见下表 1。 此外， 图 3显示了对照例中所得的 RNA片段大小的 Agilent Bioanalyzer 2100检测图， 其中 RNA片段来自 MAQC-Agilent, 利用 Illumina测序平台提供的 5xRNA 断裂试剂在 94°C断裂 5min所得。  After completion of the reaction, the PCR product was purified by QIAquick PCR Purification Kit (Qiagen), dissolved in 32 μl of the eluate, and the purified product was detected and sequenced using Agilent Bioanalyzer 2100 and Illumina Hiseq 2000. Among them, the sequencing results are shown in Table 1 below. In addition, Figure 3 shows the Agilent Bioanalyzer 2100 assay of the RNA fragment size obtained in the control, in which the RNA fragment was obtained from MAQC-Agilent using a 5x RNA cleavage reagent provided by the Illumina sequencing platform at 94 °C for 5 min.
如实施例 1和对照例所述，对两种^:阵列质量控制标准品（ MAQC标准品） MAQC-AB 和 MAQC-Agilent分别利用对照例 Illumina测序平台提供的方法 a和本发明的方法 b (实施 例 1的方法） 同时构建文库， 并各做三个重复（1、 2和 3 ), 然后利用 Illumina HiSeq2000 测序仪对获得的样本 MAQC-AB和 MAQC-Agilent的各 RNA测序文库进行测序，并对测序 数据进行分析，以便明确本发明的构建 RNA测序文库的方法对所得文库的测序数据的影响。 其中， 利用本发明的构建 RNA测序文库的方法 b (实施例 1的方法） 以及 Illumina测序平 台提供的方法 a (对照例的方法）构建的样本 MAQC-AB和 MAQC-Agilent的各 RNA测序 文库的测序数据及其分析结果见下表 1-3。 其中， 对利用 Illumina测序平台提供的方法 a以 及本发明的方法 b所得的 RNA测序文库的测序数据的分析包括三方面：测序原始数据统计、 与 QPCR的绝对定量的基因表达相关性分析以及相同数据量检测基因数的比较分析。 表 1 显示了 a、 b两种方法构建的 MAQC标准品文库的原始测序数据统计，从表 1中看出对同一 样品， a、 b两种的建库方法得到的 reads比对到基因上的比率相当， 正负偏差小于 5% , 可 以认为两种方法并无显著差别。 表 2显示了 a、 b两种建库方法所得到的测序数据中基因表 达量与 QPCR检测结果的相关性分析结果。 具体地， 通过将两种不同建库方法得到的基因 表达量与 QPCR检测同样样品得到的基因表达量进行 Spearman相关性分析获得相关性分析 结果， 其中相关系数从 0-1 , 数字越大， 说明两者之间越接近， 即相关性越好。 从表 2中数 据看， a、 b两种方法的相关系数都达到 0.85以上（大于 0.8, 符合标准）， 说明本发明方法 b的 RNA测序与 QPCR的相关性高， 定量准确； 两种方法得到的绝对定量的基因表达的相 关系数非常接近，表明和 Illumina提供的方法 a的数据相比，本发明的方法 b所得的数据可 靠、 可信。 表 3显示了 a、 b两种建库方法所得的测序相同数据量中检测到的基因数的比较 结果。 其中， 同样的数据量， 基因数越接近， 表明两种方法结果越相似。 表 3 中的数据显 示， 对同一样品， 两种不同的建库方法在相同数据量检测到的基因数相差无几， 由此证实 本发明的建库方法 b与 Illumina提供的方法 a得到的数据一致， 从而证明本发明的 RNA断 裂试剂及其打断条件适用于 mRNA-seq建库， 得到的数据真实、 可信， 且对信息分析没有 影响。 As described in Example 1 and the comparative examples, the two methods: array quality control standards (MAQC standards) MAQC-AB and MAQC-Agilent were respectively subjected to the method a provided by the comparative Illumina sequencing platform and the method b of the present invention ( Method of Example 1) Simultaneously construct a library and make three replicates (1, 2, and 3), respectively, and then utilize Illumina HiSeq2000 The sequencer was used to sequence each of the obtained RNA sequencing libraries of samples MAQC-AB and MAQC-Agilent, and the sequencing data was analyzed to determine the influence of the method of constructing the RNA sequencing library of the present invention on the sequencing data of the obtained library. Wherein, each of the RNA sequencing libraries of the samples MAQC-AB and MAQC-Agilent constructed by the method b of constructing an RNA sequencing library of the present invention (the method of Example 1) and the method a (the method of the comparative example) provided by the Illumina sequencing platform are used. The sequencing data and its analysis results are shown in Table 1-3 below. Among them, the analysis of the sequencing data of the RNA sequencing library obtained by the method a provided by the Illumina sequencing platform and the method b of the present invention includes three aspects: sequencing raw data statistics, absolute quantitative gene expression correlation analysis with QPCR, and the same data. Comparative analysis of the number of detected genes. Table 1 shows the original sequencing data of the MAQC standard library constructed by the two methods a and b. It can be seen from Table 1 that the reads of the same sample, a and b are compared to the gene. The ratio is equivalent, and the positive and negative deviations are less than 5%. It can be considered that there is no significant difference between the two methods. Table 2 shows the correlation analysis between the gene expression amount and the QPCR detection results in the sequencing data obtained by the two methods of database construction a and b. Specifically, the correlation analysis results are obtained by performing Spearman correlation analysis on the gene expression amount obtained by two different database construction methods and the same sample obtained by QPCR detection, wherein the correlation coefficient is from 0-1, and the number is larger, indicating The closer the two are, the better the correlation. From the data in Table 2, the correlation coefficients of both a and b methods are above 0.85 (greater than 0.8, in line with the standard), indicating that the RNA sequencing of the method b of the present invention is highly correlated with QPCR, and the quantification is accurate; The correlation coefficients for absolute quantitative gene expression are very close, indicating that the data obtained by method b of the present invention is reliable and reliable compared to the data for method a provided by Illumina. Table 3 shows the comparison of the number of genes detected in the same amount of data obtained by sequencing the two methods of a and b. Among them, the same amount of data, the closer the number of genes, the more similar the results of the two methods. The data in Table 3 shows that for the same sample, the number of genes detected by the same data amount is similar for the two different database construction methods, thus confirming that the database construction method b of the present invention is consistent with the data obtained by the method a provided by Illumina. Thus, it was confirmed that the RNA fragmentation reagent of the present invention and its breaking conditions are suitable for mRNA-seq construction, and the obtained data is true and reliable, and has no influence on information analysis.
a、 b两种方法构建的 MAQC标准品文库的原始测序数据统计  The original sequencing data of the MAQC standard library constructed by a and b methods
Figure imgf000012_0001
表 2: a、 b方法构建的 MAQC标准品文库的基因表达量与 QPCR检测结果的相关性分析
Figure imgf000012_0001
Table 2: Correlation analysis between gene expression levels of MAQC standard library constructed by a and b methods and QPCR detection results
Figure imgf000013_0001
较
Figure imgf000013_0001
More
Figure imgf000013_0002
以下实施例 2~10是以同样的 RNA样品（ MAQC-AB或 MAQC-Agilent )在固定打断时 间为 10min、打断温度为 94 °C的情况下，检测所述 RNA断裂试剂组分和浓度的调整对 RNA 断裂效果影响的实例。
Figure imgf000013_0002
In the following Examples 2 to 10, the same RNA sample (MAQC-AB or MAQC-Agilent) was used to detect the RNA fragmentation reagent component and concentration with a fixed breaking time of 10 min and an interrupting temperature of 94 °C. An example of the effect of adjustment on the effect of RNA fragmentation.
实施例 2:  Example 2:
2.1样品试剂  2.1 sample reagent
3x RNA断裂试剂：  3x RNA Breaking Reagents:
lOOmM Tris-HCl ( pH 8.0 ), 400mM KCl, 8mM MgCl2, lOOmM ZnCl2 (断裂试剂的各 组分均来自 Sigma, 试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实施例 1。 lOOmM Tris-HCl (pH 8.0), 400 mM KCl, 8 mM MgCl 2 , 100 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent does not contain RNase); the source of other sample and reagent buffer is the same as in Example 1 .
2.2实验步骤  2.2 Experimental steps
实验步骤同实施例 1中的步骤 1.2.1 1.2.7。 实施例 3:  The experimental procedure is the same as step 1.2.1 1.2.7 in Example 1. Example 3:
3.1样品试剂 3x RNA断裂试剂： 350mM Tris-HCl ( H 8.0 ), 80mM NaCl, lOOmM MgCl2, 8mM ZnCl2 (断裂试剂的各组分均来自 Sigma,试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实 施例 1。 3.1 sample reagent 3x RNA fragmentation reagent: 350 mM Tris-HCl (H 8.0 ), 80 mM NaCl, 100 mM MgCl 2 , 8 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); other sample and reagent buffers The source is the same as in Example 1.
3.2实险步骤  3.2 actual risk steps
实险步骤同实施例 1中的步骤 1.2.1 1.2.7。 实施例 4:  The actual risk steps are the same as those in Example 1 1.2.1 1.2.7. Example 4:
4.1样品试剂  4.1 sample reagent
3x RNA断裂试剂： 150mM Tris-HCl ( pH 8.0 ), 300mM KC1, 90mM NaCl, 80mM MgCl2 (断裂试剂的各组分均来自 Sigma,试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实 施例 1。 3x RNA fragmentation reagent: 150 mM Tris-HCl (pH 8.0), 300 mM KC1, 90 mM NaCl, 80 mM MgCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); source of other sample and reagent buffers Same as Example 1.
4.2实验步骤  4.2 Experimental steps
实验步骤同实施例 1中的步骤 1.2.1 1.2.7。 实施例 5:  The experimental procedure is the same as step 1.2.1 1.2.7 in Example 1. Example 5
5.1样品试剂  5.1 sample reagent
3x RNA断裂试剂： 300mM Tris-HCl ( pH 8.0 ), 250mM KC1, lOOmM NaCl, lOmM ZnCl2 3x RNA fragmentation reagent: 300 mM Tris-HCl (pH 8.0), 250 mM KC1, 100 mM NaCl, 10 mM ZnCl 2
(断裂试剂的各组分均来自 Sigma,试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实 施例 1。 (The components of the fragmentation reagent are all from Sigma and the reagents are free of RNase); the source of the other sample and reagent buffers is the same as in Example 1.
5.2实验步骤  5.2 Experimental steps
实验步骤同实施例 1中的步骤 1.2.1~1.2.7。 实施例 6:  The experimental procedure is the same as steps 1.2.1 to 1.2.7 in Example 1. Example 6:
6.1样品试剂  6.1 sample reagent
3x RNA断裂试剂： 250mM Tris-HCl( pH 8.0 ), lOOmM KC1, lOOmM NaCl, 60mM MgCl2,3x RNA fragmentation reagent: 250 mM Tris-HCl (pH 8.0), 100 mM KC1, 100 mM NaCl, 60 mM MgCl 2 ,
15mM ZnCl2 (断裂试剂各组分均来自 Sigma, 试剂不含 RNA酶）； 其它样品和试剂緩冲液 的来源同实施例 1。 15 mM ZnCl 2 (all components of the fragmentation reagent were from Sigma, the reagent contained no RNase); the source of the other sample and reagent buffer was the same as in Example 1.
6.2实验步骤  6.2 Experimental steps
实验步骤同实施例 1中的步骤 1.2.1 1.2.7。 实施例 7:  The experimental procedure is the same as step 1.2.1 1.2.7 in Example 1. Example 7
7.1样品试剂  7.1 sample reagent
3x RNA断裂试剂： 200mM Tris-HCl ( pH 8.0 ), 90mM NaCl, lOOmM MgCl2 (断裂试 剂的各组分均来自 Sigma, 试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实施例 1。 3x RNA fragmentation reagent: 200 mM Tris-HCl (pH 8.0), 90 mM NaCl, 100 mM MgCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); the source of other sample and reagent buffers is the same as the example 1.
7.2实验步骤  7.2 Experimental steps
实验步骤同实施例 1中的步骤 1.2.1~1.2.7。 实施例 8: The experimental procedure is the same as steps 1.2.1 to 1.2.7 in Example 1. Example 8
8.1样品试剂  8.1 sample reagent
3xRNA断裂试剂： lOOmM Tris-HCl ( pH8.0 ), 250mM NaCl, 8mM ZnCl2 (断裂试剂的 各组分均来自 Sigma, 试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实施例 1。 3xRNA cleavage reagent: lOOmM Tris-HCl (pH 8.0), 250 mM NaCl, 8 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent does not contain RNase); the source of other sample and reagent buffer is the same as the example 1.
8.2实险步骤  8.2 real risk steps
实验步骤同实施例 1中的步骤 1.2.1 1.2.7。 实施例 9:  The experimental procedure is the same as step 1.2.1 1.2.7 in Example 1. Example 9
9.1样品试剂  9.1 sample reagent
3xRNA断裂试剂： 200mM Tris-HCl ( H 8.0 ), 300mM KC1, 80mM MgCl2 (断裂试剂 各组分来自 Sigma, 试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实施例 1。 3xRNA cleavage reagent: 200 mM Tris-HCl (H 8.0 ), 300 mM KC1, 80 mM MgCl 2 (the components of the fragmentation reagent were from Sigma, the reagent contained no RNase); the source of the other sample and reagent buffer was the same as in Example 1.
9.2实险步骤  9.2 real risk steps
实验步骤同实施例 1中的步骤 1.2.1 1.2.7。 实施例 10:  The experimental procedure is the same as step 1.2.1 1.2.7 in Example 1. Example 10
10.1样品试剂  10.1 sample reagent
3xRNA断裂试剂： 200mM Tris-HCl ( pH 8.0 ), lOOmM KC1, 15mM ZnCl2 (断裂试剂 的各组分均来自 Sigma, 试剂不含 RNA酶）； 其它样品和试剂緩冲液的来源同实施例 1。 3xRNA cleavage reagent: 200 mM Tris-HCl (pH 8.0), 100 mM KC1, 15 mM ZnCl 2 (each component of the fragmentation reagent is from Sigma, the reagent contains no RNase); the source of other sample and reagent buffer is the same as in Example 1 .
10.2实验步骤  10.2 Experimental steps
实验步骤同实施例 1中的步骤 1.2.1 1.2.7。 对实施例 2~10 所得的 RNA-Seq文库（ MAQC-AB 或 MAQC- Agilent )进行 Agilent Bioanalyzer 2100检测。检测图（未列出）显示对同样的样本，在同样的断裂条件 94 °C lOmin 下， 这 9个调整 3xRNA断裂试剂的组分组成及浓度的方案对 RNA的打断效果不如或与实 施例 1的 3xRNA断裂试剂（ 200mM Tris-HCl ( pH 8.0 ), lOOmM KC1, 15mM MgCl2, 15mM ZnCl2 ) 的效果相近。 The experimental procedure is the same as step 1.2.1 1.2.7 in Example 1. The RNA-Seq library (MAQC-AB or MAQC-Agilent) obtained in Examples 2 to 10 was subjected to Agilent Bioanalyzer 2100 detection. The test pattern (not shown) shows that for the same sample, under the same rupture condition of 94 °C lOmin, the composition of the 9 components and concentration adjustment of the 3xRNA cleavage reagent is not as good as the RNA interrupting effect or with the examples. The effect of 1 3x RNA fragmentation reagent (200 mM Tris-HCl (pH 8.0), 100 mM KC1, 15 mM MgCl 2 , 15 mM ZnCl 2 ) was similar.
本领域的技术人员可以了解， 只要将维持 pH值在 7至 9之间的生物緩冲液， 用于第一 链合成的一价金属离子和用于断裂 RNA的二价金属离子这三种组分混合在一起， 并调配这 三种组分在有效浓度， 就能够达到本发明的基本目的。 为达到更好的效果， 本发明也相应 给出了这三种组分的优选组合和浓度： 生物緩冲液优选为 Tris 緩冲液， 其浓度为 100-350mM, 优选 150-300mM, 更优选 200-250mM, 且将 RNA断裂试剂 pH值维持在 7 至 9之间；一价金属离子优选为 K+和/或 Na+,更优选 K+,—价金属离子的浓度为 80-400mM, 优选 100-300mM,更优选 200-250mM。二价金属阳离子优选为 Mg2+和 /或 Zn2+,更优选 Mg2+ 和 Zn2+, 其中 Mg2+离子的浓度为 8-100mM, 优选 10-80mM, 更优选 15-60mM, Zn2+离子 的浓度为 8-100mM, 优选 10-80mM, 更优选 15-60mM。 使用这些参数或者在这些参数的附 近的 RNA断裂试剂， 本领域的技术人员将很容易的达到本发明的目的， 即简化操作步骤， 节约时间， 降低成本和实现自动化。 实施例 11: Those skilled in the art will appreciate that as long as the biological buffer having a pH between 7 and 9 is maintained, the monovalent metal ion for the first strand synthesis and the divalent metal ion for the cleavage RNA are three groups. The basic purpose of the present invention can be attained by mixing together and blending the three components at an effective concentration. In order to achieve a better effect, the present invention also correspondingly gives a preferred combination and concentration of the three components: The biological buffer is preferably a Tris buffer at a concentration of 100-350 mM, preferably 150-300 mM, more preferably 200-250 mM, and maintaining the pH of the RNA fragmentation reagent between 7 and 9; the monovalent metal ion is preferably K+ and/or Na + , more preferably K+, and the concentration of the valence metal ion is 80-400 mM, preferably 100- 300 mM, more preferably 200-250 mM. The divalent metal cation is preferably Mg 2+ and/or Zn 2+ , more preferably Mg 2+ and Zn 2+ , wherein the concentration of Mg 2+ ions is 8-100 mM, preferably 10-80 mM, more preferably 15-60 mM, Zn The concentration of 2+ ions is 8-100 mM, preferably 10-80 mM, more preferably 15-60 mM. Use these parameters or attach them to these parameters For near RNA fragmentation reagents, those skilled in the art will readily achieve the objects of the present invention, namely, simplifying the steps of operation, saving time, reducing costs and automating. Example 11
为了验证本发明的稳定性和可重复性， 在相同的断裂试剂及作用条件下 （ 3xRNA断裂 试剂： 200mM Tris-HCl ( ρΗ 8.0 ), lOOmM KC1, 15mM MgCl2, 15mM ZnCl2, 94 °C lOmin ), 选取不同物种进行平行建库，所选样品除了 MAQC- Agilent和 MAQC-AB ,还有利用 TRIzol ( Invitrogen )提取的小鼠、 水稻、 玉米或真菌的 RNA。 In order to verify the stability and reproducibility of the present invention, under the same cleavage reagent and action conditions (3xRNA cleavage reagent: 200 mM Tris-HCl (ρΗ 8.0), 100 mM KC1, 15 mM MgCl 2 , 15 mM ZnCl 2 , 94 ° C lOmin In addition to MAQC-Agilent and MAQC-AB, RNAs from mice, rice, maize or fungi extracted with TRIzol (Invitrogen) were selected for parallel construction.
11.1样品试剂  11.1 sample reagent
MAQC- Agilent, MAQC-AB , 以及利用 TRIzol ( Invitrogen )提取的小鼠、 水稻、 玉米 或真菌的 RNA; 各试剂緩冲液， 同实施例 1。  MAQC-Agilent, MAQC-AB, and RNA of mouse, rice, maize or fungi extracted using TRIzol (Invitrogen); each reagent buffer, same as in Example 1.
11.2实险步骤  11.2 Reality steps
对上述不同样品进行平行建库， 实验步骤同实施例 1中的步骤 1.2.1 1.2.7 , 以便分别将 各样品及其平行样断裂为 RNA片段。  The above different samples were constructed in parallel, and the experimental procedure was the same as the steps 1.2.1 and 1.2.7 in Example 1 to respectively break each sample and its parallel sample into RNA fragments.
利用 Agilent Bioanalyzer 2100分别检测获得的各样品及其平行样的 RNA片段大小， 结 果见图 7-18。 图 7-18 分别显示了本实施例获得的各样品及其平行样的 RNA 片段大小的 Agilent Bioanalyzer 2100检测图。 其中， 图 7和图 8中的 RNA片段分别来自 MAQC-AB及 其平行样； 图 9和图 10的 RNA片段分别来自 MAQC- Agilent及其平行样； 图 11和图 12 的 RNA片段分别来自玉米 RNA及其平行样；图 13和图 14的 RNA片段分别来自水稻 RNA 及其平行样； 图 15和图 16的 RNA片段分别来自真菌 RN A及其平行样； 图 17和图 18的 RNA片段分别来自小鼠 RNA及其平行样。 从每组样品的两个重复来看， 本发明的 RNA断 裂试剂及其打断条件对同一样品的重复性非常好， 打断片段与集中度都很相似， 如图 9和 图 10显示， 获得的 MAQC-Agilent及其平行样的打断片段非常接近， 峰图也非常相似。 由 此， 我们认为本发明的 RNA断裂试剂及打断 RNA的方法对同一样品的重复性良好； 在不 同样品之间， 由于样品的特殊性， 打断的片段大小有些许差别， 但峰形都很正常， 片段集 中度都较高， 覆盖度也很接近， 因此， 本发明的 RNA断裂试剂及打断 RNA的方法适用于 所测试的样品， 打断效果良好， 具有良好的可重复性与稳定性。 工业实用性  The size of the obtained RNA fragments of each sample and its parallel samples was measured by Agilent Bioanalyzer 2100, and the results are shown in Figures 7-18. Figure 7-18 shows the Agilent Bioanalyzer 2100 test chart for the size of each sample and its parallel sample RNA fragments obtained in this example. Among them, the RNA fragments in Figures 7 and 8 are from MAQC-AB and their parallel samples, respectively; the RNA fragments in Figures 9 and 10 are from MAQC-Agilent and their parallel samples, respectively; the RNA fragments in Figure 11 and Figure 12 are from corn, respectively. RNA and its parallel samples; the RNA fragments of Figure 13 and Figure 14 are from rice RNA and their parallel samples, respectively; the RNA fragments of Figure 15 and Figure 16 are from the fungal RN A and their parallel samples, respectively; the RNA fragments of Figure 17 and Figure 18, respectively From mouse RNA and its parallel samples. From the two repetitions of each set of samples, the RNA fragmentation reagent of the present invention and its breaking conditions are very reproducible to the same sample, and the interrupted fragments are very similar to the concentration, as shown in Fig. 9 and Fig. 10, The MAQC-Agilent and its parallel-like interrupted fragments are very close, and the peak maps are very similar. Therefore, we believe that the RNA fragmentation reagent of the present invention and the method for interrupting RNA have good repeatability to the same sample; between different samples, due to the particularity of the sample, the size of the fragment is slightly different, but the peak shape is It is normal, the fragment concentration is high, and the coverage is very close. Therefore, the RNA fragmentation reagent and the method for interrupting RNA of the present invention are suitable for the sample to be tested, have good breaking effect, and have good repeatability and stability. Sex. Industrial applicability
本发明的 RNA断裂试剂、 打断 RNA的方法、 构建 RNA测序文库的方法、 RNA测序 文库以及测序方法， 能够有效地应用于样本的 RNA测序文库的构建以及测序， 并且获得的 文库质量好， 测序结果准确。  The RNA fragmentation reagent, the method for interrupting RNA, the method for constructing an RNA sequencing library, the RNA sequencing library and the sequencing method of the invention can be effectively applied to the construction and sequencing of the RNA sequencing library of the sample, and the obtained library is of good quality and sequenced. The result is accurate.
尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。  Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
在本说明书的描述中， 参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。 In the description of the present specification, the terms "one embodiment", "some embodiments", "illustrative embodiments", "show" The description of the ",""specificexamples", or "some examples" and the like is intended to mean that the specific features, structures, materials, or characteristics described in connection with the embodiments or examples are included in the at least one embodiment or example. The above description of the terminology does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be in a suitable manner in any one or more embodiments or examples. Combine.

Claims

权利要求书 Claim
1、 一种 RNA断裂试剂， 其特征在于， 包括：  1. An RNA fragmentation reagent, comprising:
生物緩冲液， 所述生物緩冲液适于将所述 RNA断裂试剂的 pH值维持在 7~9之间； 一价金属离子和二价金属离子；  a biological buffer, wherein the biological buffer is adapted to maintain a pH of the RNA fragmentation reagent between 7 and 9; a monovalent metal ion and a divalent metal ion;
其中， 所述生物緩冲液、 一价金属离子和二价金属离子均处于有效浓度。  Wherein, the biological buffer, the monovalent metal ion and the divalent metal ion are all at an effective concentration.
2、 权利要求 1所述的 RNA断裂试剂， 其特征在于， 所述生物緩冲液为 Tris緩冲液。 The RNA fragmentation reagent according to claim 1, wherein the biological buffer is a Tris buffer.
3、 根据权利要求 2所述的 RNA断裂试剂， 其特征在于， 所述 Tris緩冲液的浓度为 100-350mM。 The RNA fragmentation reagent according to claim 2, wherein the concentration of the Tris buffer is 100-350 mM.
4、 根据权利要求 2所述的 RNA断裂试剂， 其特征在于， 所述 Tris緩冲液的浓度为 The RNA fragmentation reagent according to claim 2, wherein the concentration of the Tris buffer is
150-300mM。 150-300 mM.
5、 根据权利要求 2所述的 RNA断裂试剂， 其特征在于， 所述 Tris緩冲液的浓度为 200-250mM。  The RNA fragmentation reagent according to claim 2, wherein the concentration of the Tris buffer is 200-250 mM.
6、 根据权利要求 2所述的 RNA断裂试剂， 其特征在于， 所述 Tris緩冲液将所述 RNA 断裂试剂的 pH维持在 7至 9之间。  The RNA fragmentation reagent according to claim 2, wherein the Tris buffer maintains the pH of the RNA fragmentation reagent between 7 and 9.
7、根据权利要求 1所述的 RNA断裂试剂，其特征在于，所述一价金属离子为 K+和 Na+ 的至少一种。 The RNA fragmentation reagent according to claim 1, wherein the monovalent metal ion is at least one of K+ and Na + .
8、 根据权利要求 7所述的 RNA断裂试剂， 其特征在于， 所述一价金属离子为 K+。The RNA fragmentation reagent according to claim 7, wherein the monovalent metal ion is K + .
9、 根据权利要求 7所述的 RNA断裂试剂， 其特征在于， 所述一价金属离子的浓度为 80-400mM。 The RNA fragmentation reagent according to claim 7, wherein the monovalent metal ion has a concentration of 80 to 400 mM.
10、 根据权利要求 7所述的 RNA断裂试剂， 其特征在于， 所述一价金属离子的浓度为 90-300mM。  The RNA fragmentation reagent according to claim 7, wherein the monovalent metal ion has a concentration of 90 to 300 mM.
11、 根据权利要求 7所述的 RNA断裂试剂， 其特征在于， 所述一价金属离子的浓度为 100-250mM。  The RNA fragmentation reagent according to claim 7, wherein the concentration of the monovalent metal ion is from 100 to 250 mM.
12、 根据权利要求 1所述的 RNA断裂试剂 其特征在于， 所述二价金属离子为 Mg: 和 Zn2+的至少一种。 The RNA fragmentation reagent according to claim 1, wherein the divalent metal ion is at least one of Mg : and Zn 2+ .
13、 根据权利要求 1所述的 RNA断裂试剂 其特征在于， 所述二价金属离子为 Mg: 和 Zn2+。 The RNA fragmentation reagent according to claim 1, wherein the divalent metal ion is Mg : and Zn 2+ .
14、 根据权利要求 13所述的 RNA断裂试剂 其特征在于 Mg2+的浓度为 8-100mM。 14. The RNA fragmentation reagent according to claim 13, characterized in that the concentration of Mg 2+ is from 8 to 100 mM.
15、 根据权利要求 13所述的 RNA断裂试剂 其特征在于 Mg2+的浓度为 10-80mM。15. The RNA fragmentation reagent according to claim 13, characterized in that the concentration of Mg 2+ is from 10 to 80 mM.
16、 根据权利要求 13所述的 RNA断裂试剂 其特征在于 Mg2+的浓度为 15-60mM。16. The RNA fragmentation reagent according to claim 13, characterized in that the concentration of Mg 2+ is from 15 to 60 mM.
17、 根据权利要求 13所述的 RNA断裂试剂 其特征在于 Zn2+的浓度为 8-100mM。17. The RNA fragmentation reagent according to claim 13, characterized in that the concentration of Zn 2+ is from 8 to 100 mM.
18、 根据权利要求 13所述的 RNA断裂试剂 其特征在于 Zn2+的浓度为 10-80mM。18. The RNA fragmentation reagent according to claim 13, characterized in that the concentration of Zn 2+ is from 10 to 80 mM.
19、 根据权利要求 13所述的 RNA断裂试剂 其特征在于 Zn2+的浓度为 15-60 mM。 19. The RNA fragmentation reagent according to claim 13, characterized in that the concentration of Zn 2+ is from 15 to 60 mM.
20、 根据权利要求 1~19任一项所述的 RNA断裂试剂， 其特征在于， 所述 RNA来源于 原核或真核生物。 The RNA fragmentation reagent according to any one of claims 1 to 19, wherein the RNA is derived from a prokaryotic or eukaryotic organism.
21、 权利要求 1~20任一项所述的 RNA断裂试剂在打断 RNA中的应用。 The use of the RNA fragmentation reagent according to any one of claims 1 to 20 for interrupting RNA.
22、 一种打断 RNA的方法， 其特征在于， 包括：  22. A method of interrupting RNA, comprising:
使用权利要求 1~20任一项所述的 RNA断裂试剂打断 RNA。  The RNA is cleaved by the RNA fragmentation reagent according to any one of claims 1 to 20.
23、 一种构建 RNA测序文库的方法， 包括以下步骤：  23. A method of constructing an RNA sequencing library, comprising the steps of:
( a )使用权利要求 1~20任一项所述的 RNA断裂试剂打断 RNA, 以便获得 RNA片段； (a) interrupting RNA by using the RNA fragmentation reagent according to any one of claims 1 to 20 to obtain an RNA fragment;
( b )在步骤（ a )后的反应体系中直接加入逆转录酶和随机引物， 进行逆转录， 以便合 成 cDNA第一链； (b) directly adding a reverse transcriptase and a random primer to the reaction system after the step (a), and performing reverse transcription to synthesize the first strand of the cDNA;
( c )在步骤（ b )后的反应体系中加入聚合酶和 cDNA二链合成緩冲液，以便合成 cDNA 第二链并获得双链 cDNA;  (c) adding a polymerase and a cDNA double-strand synthesis buffer to the reaction system after the step (b) to synthesize the second strand of the cDNA and obtain a double-stranded cDNA;
( d ) 纯化所述双链 cDNA;  (d) purifying the double-stranded cDNA;
( e )对所述双链 cDNA进行末端修复， 末端添加碱基 "A" 和连接测序接头， 以便得 到连接产物； 以及  (e) end-repairing the double-stranded cDNA, adding a base "A" at the end and ligating a sequencing link to obtain a ligation product;
( f )对所述连接产物进行 PCR扩增， 以便获得扩增产物，所述扩增产物构成所述 RNA 测序文库。  (f) PCR-amplifying the ligation product to obtain an amplification product, the amplification product constituting the RNA sequencing library.
24、根据权利要求 23所述的方法 , 其特征在于， 在 70-100 °C的温度下， 使用所述 RNA 断裂试剂打断 RNA。  The method according to claim 23, wherein the RNA is cleaved by using the RNA fragmentation reagent at a temperature of 70 to 100 °C.
25、 根据权利要求 23所述的方法 , 其特征在于， 在 85-95 °C的温度下， 使用所述 RNA 断裂试剂打断 RNA。  25. The method according to claim 23, wherein the RNA is cleaved by using the RNA fragmentation reagent at a temperature of 85-95 °C.
26、 根据权利要求 23所述的方法 , 其特征在于， 在 94 °C的温度下， 使用所述 RNA断 裂试剂打断 RNA。  26. The method according to claim 23, wherein the RNA is interrupted by using the RNA fragmentation reagent at a temperature of 94 °C.
27、 根据权利要求 23所述的方法 , 其特征在于， 使用所述 RNA断裂试剂处理 RNA的 作用时间是 3-12min。  27. The method according to claim 23, wherein the RNA treatment time using the RNA fragmentation reagent is 3-12 min.
28、 根据权利要求 23所述的方法 , 其特征在于， 使用所述 RNA断裂试剂处理 RNA的 作用时间是 6-10min。  28. The method according to claim 23, wherein the RNA treatment time using the RNA fragmentation reagent is 6-10 min.
29、 根据权利要求 23所述的方法 , 其特征在于， 使用所述 RNA断裂试剂处理 RNA的 作用时间是 10min。  29. The method according to claim 23, wherein the RNA treatment time using the RNA fragmentation reagent is 10 min.
30、 权利要求 23所述的方法， 其特征在于， 所述 RNA片段的长度为 60-1500nt。  30. The method of claim 23, wherein the RNA fragment is between 60 and 1500 nt in length.
31、 根据权利要求 23所述的方法， 其特征在于， 所述 RNA片段的长度为 150-700nt。 31. The method of claim 23, wherein the RNA fragment is between 150 and 700 nt in length.
32、 根据权利要求 23所述的方法， 其特征在于， 所述 RNA片段的长度为 150-500nt。 32. The method of claim 23, wherein the RNA fragment is between 150 and 500 nt in length.
33、 一种 RNA测序文库， 其是由根据权利要求 23-32中任一项所述的方法构建的。33. An RNA sequencing library constructed by the method of any one of claims 23-32.
34、 一种测序方法， 其特征在于： 34. A sequencing method characterized by:
根据权利要求 23-32中任一项所述的方法构建 RNA测序文库； 以及  Constructing an RNA sequencing library according to the method of any one of claims 23-32;
对所述 RNA测序文库进行测序，  Sequencing the RNA sequencing library,
其巾，  Its towel,
所述测序使用高通量测序平台进行， 所述高通量测序平台为选自 Illumina/Solexa、 ABI The sequencing is performed using a high throughput sequencing platform selected from Illumina/Solexa, ABI
Solid和 Roche 454测序平台的至少一种。 At least one of the Solid and Roche 454 sequencing platforms.
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