A method and kit for constructing a ribosome RNA sequencing library

Abstract

The application discloses a ribosome RNA sequencing library construction method and kit, and relates to the technical field of biology. The method comprises the following steps: 1) total RNA breaking and ribosome RNA rapid hybridization and RNA template 1st strand cDNA synthesis; 2) 2nd strand cDNA synthesis; 3) double-stranded cDNA 3' end A addition and adaptor ligation; and 4) dUTP-containing template strand removal and library amplification. The application adopts special rRNA depletion probes to realize one-step rapid rRNA removal and complete library construction, greatly reduces operation steps in the rRNA depletion library construction and shortens reaction time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene sequencing technology, and particularly relates to a method for constructing a sequencing library of a ribosome RNA sequencing library and a kit. BACKGROUND

[0002] The ribosome RNA removal in RNA-seq mainly includes three methods, the first method is polyA RNA capture by oligo dT magnetic beads, the second method is rRNA removal by enzymatic method, and the third method is rRNA removal by streptomycin magnetic beads method; the three methods are either relatively cumbersome in the operation process or relatively long in the reaction time, and especially the polyA RNA capture by oligo dT magnetic beads has a relatively high requirement on the quality of the RNA sample.

[0003] Therefore, it is necessary to study a method for quickly removing rRNA and completing library construction, so as to reduce the operation steps, shorten the reaction time, and better promote the application. SUMMARY

[0004] In view of the defects of the prior art, the present application provides a method for constructing a ribosome RNA sequencing library and a kit, which realizes one-step rRNA removal to effectively simplify the library construction process.

[0005] In order to achieve the above purpose, the present application provides a method for constructing a ribosome RNA sequencing library, comprising the following steps:

[0006] 1. Fragmenting total RNA, then hybridizing rRNA with anti rRNA probe to form hybrid chains of rRNA and single-stranded DNA; performing chain-specific reverse transcription on the RNA template which is not hybridized by random primers to form 1st cDNA, forming hybrid chains of RNA and 1st cDNA; the anti rRNA probe is modified with dU in the middle;

[0007] 2. Replacing the rRNA chain and the RNA template in S1 with 2nd cDNA containing dUTP, both forming double-stranded cDNA;

[0008] 3. Performing end repair and adding A at the 3' end, and connecting adapters for high-throughput sequencing at both ends;

[0009] 4. Removing the template chain containing dUTP, performing PCR amplification of the library, and obtaining the sequencing library by magnetic bead purification.

[0010] Further, two ends of the Anti rRNA are closed modification; preferably, the 5' end of the Anti rRNA is closed modification of amino modification, and the 3' end is closed modification of C6 Spacer modification, C3 Spacer modification or amino modification plus C6 Spacer modification.

[0011] Further, the Anti rRNA probe is less than 100 bp in length.

[0012] Further, the Total RNA is fragmented by Mg 2+ The RNA is fragmented, and the reaction system of the RNA fragmentation comprises Tris-HCl and KCl.

[0013] Further, the reaction system of the reverse transcription process in step 2 comprises reverse transcriptase, reverse transcription reaction buffer, reverse transcription primer and actinomycin D.

[0014] Further, the dUTP-containing template strand is removed by UDG enzyme in step 4.

[0015] Another object of the present application is to provide a ribosome RNA library construction kit, characterized by comprising the probe in the method for constructing a ribosome RNA sequencing library.

[0016] Further, the kit further comprises purified magnetic beads, an Index Primer and an RNA library construction system; the purified magnetic beads are Agencourt AMPure XP beads; the Index Primer is ABclonal RK20351; and the RNA library construction system is ABclonal RK20301, ABclonal RK20304 or ABclonal RK20353 plus ABclonal RK20222.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] 1. The ribosome RNA process provided by the present application is completed in one step and 30 minutes, which shortens the time and improves the efficiency.

[0019] 2. The method for constructing a ribosome RNA sequencing library provided by the present application omits mRNA enrichment, and the construction of the ribosome RNA sequencing library can be completed within 1 day, which shortens the library construction time. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. Other related drawings can also be obtained by those of ordinary skill in the art without creative labor on the basis of these drawings.

[0021] Figure 1 Structure diagram for anti-rRNA probe design.

[0022] Figure 2 Principle diagram for ribosome RNA library construction of the present application.

[0023] Figure 3 QPCR analysis comparison diagram of corresponding reference genes and corresponding regions for the library constructed by using 80nt anti rRNA probe mix of the present application.

[0024] Figure 4 QPCR analysis comparison diagram of corresponding reference genes and corresponding regions for the library constructed by using 40nt anti rRNA probe mix of the present application.

[0025] Figure 5 Library diagram constructed by using 80nt anti rRNA probe mix of the present application.

[0026] Figure 6 Library diagram constructed by using 40nt anti rRNA probe mix of the present application. DETAILED DESCRIPTION

[0027] The following examples are used to illustrate the present application, but do not limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0028] The purpose of the present application is to provide a ribosome RNA sequencing library construction method, a rapid ribosome RNA probe with special structure is designed according to species information, the probe is a DNA oligo probe, which has the advantages of Figure 1 A-1C shown in the modification, including intermediate dU modification, 5' end and 3' end closed modification, intermediate dU modification or nick modification prevents high-fidelity polymerase from amplifying the template in PCR; the purpose of 3' end and 5' end closed modification is to prevent linker ligation, and the closed modification group mainly includes amino modification, C3 Spacer modification and C6 Spacer modification, etc.

[0029] Ribosome depletion process is completed in one step within 30 min, and the flow chart and working principle diagram are shown in Figure 2 The general rRNA depletion process is complicated and time-consuming, generally taking about 2 h. The present application not only simplifies the operation process, but also shortens the reaction time, and has good ribosome depletion effect in the sequencing data.

[0030] Example 1. Design, synthesis and modification of anti rRNA probe

[0031] According to the mouse rRNA 18S sequence, anti rRNA 18S probe was synthesized, and anti rRNA 18S was divided into two parts (completed by Shanghai Shengong Biological Company), as shown in Table 1. The anti rRNA 18S sequence 5' end 859 nt, 80 nt length, 12 probes, the 5' end of each probe was modified with 5'-Amino, the 3' end was modified with 3`NH2 C6, and two dU modifications were added in the middle. As shown in Table 2, the anti rRNA 18S sequence 5' end 907 nt, 40 nt length, 23 probes, the 5' end of each probe was modified with 5'-Amino, the 3' end was modified with 3`NH2 C6, and two dU modifications were added in the middle. When designing the probe, longer probes will require more dU modifications, and probes of no more than 100 bp are generally selected.

[0032] After synthesis, 1xTE buffer was added to each tube, and the concentration of each probe was diluted to 100 uM.

[0033] Table 1:

[0034]

[0035]

[0036] Table 2:

[0037]

[0038]

[0039]

[0040] Experimental case 2

[0041] All reagents used in the construction of the library were from the Wuhan Abclonal Biotech Co., Ltd. Stranded mRNA-seq Lib Prep Kit for Illumina kit (ABclonal RK20301) components, which can also be used with Wuhan Abclonal Biotech Co., Ltd. Fast RNA-seq Lib Prep Kit for Illumina (ABclonal RK20304) kit components; RNA Truncated Adapter and PCR index are from the components in the Wuhan Abclonal Biotech Co., Ltd. RNA Adaptor Module 96 Index for Illumina kit (ABclonal RK20351); The DNA purification magnetic beads used in the process are from Beckman Coulter's Agencourt AMPure XP beads, which are stable and reliable in quality;

[0042] In this example, 1 ug of total RNA extracted from mouse liver tissue was used as the initial template for RNA library construction, and the main library construction process was as follows:

[0043] 1) RNA fragmentation, rRNA hybridization with probes

[0044] 2X Frag / Elute buffer and synthesized rRNA Probe Mix were prepared according to Table 3, and then RNA fragmentation and hybridization were performed according to the procedure in Table 4:

[0045] Table 3:

[0046]

[0047]

[0048] Table 4:

[0049] Temperature Time 94℃ 10 min 75℃ 2 min 70℃ 2 min 65℃ 2 min 60℃ 2 min 55℃ 2 min 37℃ 5 min 25℃ 5 min 4℃ Hold

[0050] 1st cDNA synthesis, where the RT Strand Specificity Reagent contains Actinomycin D components (Shenzhen Xingboseng Biotechnology Co., Ltd., Catalog No. 11421), which can inhibit reverse transcriptase to rapidly amplify rRNA depletion probes, inhibit the synthesis of 1st cDNA as a template to reduce non-specific amplification. The reaction system was prepared according to Table 5, and the reaction procedure was checked according to Table 6.

[0051] Table 5:

[0052]

[0053]

[0054] *First Strand Synthesis Enzyme Mix contains ABScript III Reverse Transcriptase ABclonal RK20408 and ABScript II Reverse Transcriptase RK21400.

[0055] In addition, the above 1st cDNA synthesis system can also be taken from Wuhan Aibote Biological Technology Co., Ltd. First Strand Synthesis Module kit component ABclona RK20353.

[0056] Table 6:

[0057] Temperature Time 25℃ 10 min 42℃ 15 min 70℃ 15 min 4℃ Hold

[0058] 2) 2nd cDNA synthesis, according to Table 7 to prepare the reaction system, the reaction procedure is 16℃, 60min.

[0059] Table 7:

[0060]

[0061] After the reaction is completed, the 2nd cDNA synthesis product is purified using magnetic beads with a volume of 2.2x reaction system, and finally the 2nd cDNA product is eluted using 37ul Low-EDTA TE.

[0062] The synthesis of 2nd cDNA is completed, that is, the ribosome removal process is completed. The above steps can be completed in 30min.

[0063] 3) End repair, according to Table 8 to prepare the reaction system for end repair and 3' end dA, the reaction procedure is: 20℃ 30min, 65℃ 30min, 4℃ Hold.

[0064] Table 8:

[0065] Reagent Volume Double-stranded cDNA 37 μL End-prep Buffer 10 μL End-prep Enzyme Mix 3 μL Total volume 50 μL

[0066] Among them, End-prep Enzyme Mix can be T4 DNA Polymerase (ABclonal RK20539), T4 Polynucleotide Kinase (ABclonal RK20524), Klenow (ABclonal RK20526), Taq Antibody (ABclonal RK20545), and one of them can be selected.

[0067] After the ligation reaction, the ligation product was purified using 0.8X ligation system volume of purification magnetic beads, and finally the ligation product was eluted using 20ul Low EDTA TE.

[0068] Table 9:

[0069] Reagent Volume End-prep DNA (Step 4.2) 50 μL Ligation Buffer 16.5 μL RNA Truncated Adapter 2.5 μL Ligase Mix 3 μL Total volume ~ 70 μL

[0070] Among them, Ligase Mix can select T4 DNA Ligase (ABclonal RK21500), and T4 DNA Ligase cannot effectively connect the template strand closed at the end.

[0071] After the ligation reaction, the ligation product was purified using 0.8X ligation system volume of purification magnetic beads, and finally the ligation product was eluted using 20ul Low EDTA TE.

[0072] UDG digestion and PCR amplification. The reaction system is prepared according to Table 10, wherein the PCR primer is taken from ABclonal RNA Adaptor Module 96Index for Illumina kit; the reaction program is as shown in Table 11.

[0073] Table 10:

[0074] Reagent Volume Ligation purified product 19.5 μL 2X PCR Mix 25 μL UDG Enzyme 0.5 μL RNA Universal Primer 2.5 μL RNA Index Primer 2.5 μL Total volume 50 μL

[0075] Among them, UDG Enzyme selects ABclonal RK20543, and Endo VIII enzyme (ABclonal RK20534) can also be added according to needs, for digestion and hydrolysis of templates containing dUTP.

[0076] Table 11:

[0077]

[0078]

[0079] Reaction was ended, and the reaction product was purified using 1.0X PCR system volume of purified magnetic beads, and finally the library was eluted using 31ul Low EDTA TE.

[0080] The above steps can achieve the construction of ribosome-free RNA library within 1 day, greatly shortening the library construction time.

[0081] Experimental Example 1

[0082] Three samples were tested in this experiment, including a control sample of 1ug Rat total RNA without adding probes for library construction, a sample of 80nt probe mix, in which 1ug Rat total RNA was added to the 80nt probe mix for library construction, and a sample of 40nt probe mix, in which 1ug Rat total RNA was added to the 40nt probe mix for library construction. The library yield is shown in Table 12.

[0083] Table 12:

[0084] Sample name Library yield (ng / ul) Probe (-) 40.8 80 nt probe mix 28 40 nt probe mxi 15.8

[0085] Qpcr quantitative detection was performed on the above three samples: mouse GapDH primers and 18s primers were used for detection, and the results are shown in Table 14. Figure 3 As can be seen from Table 14, the expression amount of the internal reference gene of the control sample without adding probes for library construction and the sample of 80nt probe mix for library construction is basically the same, and the expression amount of 18s rRNA of the sample of 80nt probe mix for library construction is significantly lower than that of the control group. Figure 4 As can be seen from Table 14, the expression amount of the internal reference gene of the control sample without adding probes for library construction and the sample of 80nt probe mix for library construction is basically the same, and the expression amount of 18s rRNA of the sample of 80nt probe mix for library construction is significantly lower than that of the control group.

[0086] Experimental Example 2

[0087] The obtained library was sequenced on the illumina platform, and the obtained data was compared with the sequences of the 5' end and 3' end of mouse RNA18s (Table 13), and it was found that the residual amount of 18s in the sample without anti rRNA probe hybridization removal was 75%, the residual amount of rRNA in the part of 80nt anti rRNA probe was 1.7%, and the residual amount of rRNA in the part of 40nt anti rRNA probe mix was 3.2%.

[0088] Table 13:

[0089] Name Insert size GC% Mapping% rRNA Rate Probe (-) 191 55 92.3 75％ Table 1 probe mix 202 54 91.1 1.7％ Table 2 probe mix 190 53 95.4 3.2％

[0090] It can be seen that the anti rRNA probes of 80 nt and 40 nt length designed based on mouse RNA 18s have significant rRNA removal effect relative to Probe(-) by using the method of the present application.

[0091] The present application can provide probes for fast rRNA removal with special structures according to species information, and different probes can be designed according to different species rRNA sequences, and the species is flexible and variable (such as human / mouse / rat, bacteria, human globin, plant), and even different anti RNA sequence probes can be designed according to different requirements, such as human globin gene.

[0092] The present application is not limited to the description and embodiments described in the specification, and therefore additional advantages and modifications can be easily realized by those skilled in the art, and the present application is not limited to specific details, representative schemes and examples of the drawings shown and described herein without departing from the spirit and scope of the general concept defined by the claims and equivalent ranges.

Claims

1. A method for constructing a ribosome RNA sequencing library, characterized by, Comprising the following steps: S1. Fragmenting total RNA, then hybridizing rRNA with anti rRNA probe to form hybridization chain of rRNA and single-stranded DNA; performing chain-specific reverse transcription on the unhybridized RNA template by random primers to form 1st cDNA, forming hybridization chain of RNA and 1st cDNA; the anti rRNA probe is modified with dU in the middle; S2. Replacing the rRNA chain and the RNA template in S1 with 2nd cDNA containing dUTP, both forming double-stranded cDNA; S3. Performing end repair and adding A at the 3' end, and connecting adapters for high-throughput sequencing at both ends; S4. Removing the template strand containing dUTP with UDG enzyme, performing PCR amplification of the library, and obtaining the sequencing library by magnetic bead purification.

2. The method for ribosome RNA sequencing library construction according to claim 1, characterized in that, The two ends of the AntirRNA are modified by blocking.

3. The method for ribosome RNA sequencing library construction according to claim 2, characterized in that, The 5' end of the AntirRNA is modified by amino modification, and the 3' end of the AntirRNA is modified by C6 Spacer modification, C3 Spacer modification, or amino modification plus C6 Spacer modification.

4. The method for ribosome RNA sequencing library construction according to claim 1, characterized in that, The AntirRNA probe is less than 100 bp in length.

5. The method for ribosome RNA sequencing library construction according to claim 1, wherein, The Total RNA is fragmented by Mg 2+ The RNA is fragmented, the fragmentation reaction comprising Tris-HCl, KCl.

6. The method for constructing a ribosome RNA- depleted sequencing library according to claim 1, wherein, The reaction system of the reverse transcription process in step S2 includes reverse transcriptase, reverse transcription reaction buffer, reverse transcription primer, and actinomycin D.

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