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CN-121975912-A - Micro RNA acetylation library-building sequencing method

CN121975912ACN 121975912 ACN121975912 ACN 121975912ACN-121975912-A

Abstract

The invention relates to a micro RNA acetylation library-building sequencing method, and belongs to the technical field of biology. Aiming at the problems of high RNA consumption and low sensitivity of the conventional acRIP-seq technology, the trace RNA acetylation library-building sequencing method provided by the invention greatly reduces the initial RNA amount required by library building from the conventional 50-500 mug to 500 ng level by optimizing the time for removing rRNA in the acRIP-seq flow (removing in advance) and improving the impurity washing mode. The optimization realizes the accurate detection of the ac4C modification level in the ng-level RNA sample, and effectively solves the application bottleneck of the technology in the detection of rare cell subsets, clinical micro samples and ac4C low-modification-abundance RNA samples.

Inventors

  • Weng Hangyou
  • HUANG HUILIN
  • HUANG FENG
  • WANG YUSHUAI
  • GAO WEIWEI

Assignees

  • 广州医科大学附属第五医院(广州再生医学与健康广东省实验室附属医院)
  • 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所）

Dates

Publication Date
20260505
Application Date
20251229

Claims (10)

  1. 1. A method for sequencing trace RNA by acetylation, library building and sequencing, which is characterized by comprising the following steps: Incubating the magnetic beads with the antibodies to obtain magnetic beads coated with the antibodies; rRNA and DNA removal, namely removing rRNA and DNA in the RNA sample by utilizing RNase H enzyme and DNase I enzyme to obtain a sample with rRNA and DNA removed; the RNA fragmentation step, namely carrying out fragmentation treatment on the sample from which rRNA and DNA are removed to obtain an immune coprecipitation reaction sample; Washing step, firstly washing the unbound antibodies on the surfaces of the magnetic beads coated with the antibodies, and then mixing the magnetic beads coated with the antibodies with an immune coprecipitation reaction sample, incubating to obtain an incubation product A, centrifuging the incubation product A to obtain an RNA-antibody-magnetic bead complex, and firstly washing the unbound immune coprecipitation reaction sample on the surfaces of the RNA-antibody-magnetic bead complex to obtain a washed RNA-antibody-magnetic bead complex, wherein the washing of the unbound immune coprecipitation reaction sample on the surfaces of the RNA-antibody-magnetic bead complex comprises washing the unbound immune coprecipitation reaction sample on the surfaces of the RNA-antibody-magnetic bead complex by using buffer solutions containing different concentrations of detergents; the elution step is that the washed RNA-antibody-magnetic bead complex is mixed with protease and then incubated to obtain an incubation product B, and the incubation product B is subjected to enzyme inactivation and elution treatment to obtain ac 4C-immunoprecipitated RNA; concentrating the ac 4C-immunoprecipitated RNA by using an ethanol precipitation method to obtain a library-building sample; library construction, namely constructing a library of library construction samples in a non-fragmentation mode to obtain a library; sequencing the library to obtain the sequencing result.
  2. 2. The method of claim 1, wherein in the step of incubating the magnetic beads with the antibody, the incubation time of the magnetic beads with the antibody is 16-30 hours; Preferably, the antibody comprises an anti-ac 4C antibody.
  3. 3. The method according to any one of claims 1 to 2, wherein in the rRNA and DNA removal step, the rRNA and DNA in the RNA sample are removed by RNase H enzyme and DNase I enzyme, which comprises the steps of pretreating the RNA sample to obtain a pretreated RNA sample, constructing a reaction system for removing rRNA by RNase H enzyme and incubating the reaction system to degrade rRNA in the pretreated RNA sample to obtain a rRNA-removed sample, constructing a reaction system for removing DNA by DNase I enzyme and incubating the reaction system to degrade DNA in the rRNA-removed sample to obtain a DNA-removed sample; Preferably, in the reaction system for removing rRNA, the volume of RNase H enzyme accounts for 9-11% of the volume of the reaction system, and in the reaction system for removing DNA, the volume of DNase I enzyme accounts for 9-11% of the volume of the reaction system; Preferably, the components of the rRNA-removing reaction system comprise 11-12 mu L, NEBNext RNase H Reaction Buffer 1.5-2.5 mu L, NEBNext Thermostable RNase H1.5-2.5 mu L and nucleic-FREE WATER 1-2 mu L of the pretreated RNA sample; Preferably, the components of the DNA removal reaction system include 19-21. Mu.L of rRNA-removed sample, 4-6. Mu.L DNase I Reaction Buffer, 2-3. Mu.L NEBNEext DNase I and 22-23. Mu.L nucleic-FREE WATER.
  4. 4. The method of any one of claims 1 to 3, wherein in the rRNA and DNA removal step, the pretreatment of the RNA sample comprises mixing the RNA sample with a probe buffer and incubating to obtain a pretreated RNA sample; Preferably, the components of the probe buffer used in the rRNA and DNA removal steps include NEBNExt v2 rRNA Depletion Solution and NEBNext Probe Hybridization Buffer.
  5. 5. The method according to any one of claims 1 to 4, wherein in the rRNA and DNA removal step, the rRNA and DNA in the RNA sample are removed by RNase H enzyme and DNase I enzyme, and the rRNA and DNA-removed sample is obtained by sequentially subjecting the DNA-removed sample to RNAClean XP bead binding to target RNA, washing to remove unbound nucleic acid fragments by ethanol, and preventing RNA degradation by mixing with RiboLock RNase inhibitor.
  6. 6. The method according to any one of claims 1 to 5, wherein the RNA fragmentation step comprises subjecting the rRNA and DNA depleted sample to non-contact sonication such that the rRNA and DNA depleted sample is cleaved into shorter fragments to obtain a co-immunoprecipitation sample.
  7. 7. The method according to any one of claims 1 to 6, wherein in the RNA fragmentation step, the non-contact ultrasonic treatment of the rRNA and DNA-removed sample comprises mixing an IP buffer containing RNase inhibitor with the rRNA and DNA-removed sample to obtain an RNA fragmentation reaction system; in the IP buffer solution containing the RNase inhibitor, the final concentration of the RNase inhibitor is 1U/mu L; Preferably, the composition of the IP buffer used in the RNA fragmentation step comprises 50 mM Tris-HCl at pH 7.5, 750 NaCl mM, 0.5% NP-40 by volume and 1U/. Mu.L RiboLock RNase inhibitor.
  8. 8. The method according to any one of claims 1 to 7, wherein washing the unbound antibody from the surface of the antibody-coated magnetic bead comprises washing the unbound antibody from the surface of the antibody-coated magnetic bead with an IP buffer, wherein the washing step comprises washing the unbound antibody from the surface of the antibody-coated magnetic bead with an IP buffer comprising 10mM pH 7.5 Tris-HCl, 250 mM NaCl, 0.1% NP-40 by volume, and 1U/. Mu. L RiboLock RNase inhibitor; In the washing step, the buffer solution containing different concentrations of detergent comprises a first RIPA buffer solution and a second RIPA buffer solution, wherein the first RIPA buffer solution comprises 10mM pH 8.0 Tris-HCl, 300mM NaCl, 1 mM EDTA, 0.5mM EGTA, 1% by volume of Triton X-100, 0.2% by volume of SDS and 0.1% by volume of sodium deoxycholate, and the second RIPA buffer solution comprises 10mM pH 8.0 Tris-HCl, 350 mM NaCl, 1 mM EDTA, 0.5mM EGTA, 1% by volume of Triton X-100, 0.23% by volume of SDS and 0.1% by volume of sodium deoxycholate; In the washing step, the non-combined co-immunoprecipitation reaction sample on the surface of the RNA-antibody-magnetic bead complex is washed by using buffer solution containing different concentrations of detergent, wherein the washing step comprises the steps of firstly washing the RNA-antibody-magnetic bead complex for 1 time by using a first RIPA buffer solution, then washing the RNA-antibody-magnetic bead complex for 2 times by using a second RIPA buffer solution, and then washing the RNA-antibody-magnetic bead complex for 1 time by using the first RIPA buffer solution; In the eluting step, the washed RNA-antibody-magnetic bead complex is mixed with protease for incubation, wherein the incubation precipitation is firstly resuspended in an eluting buffer solution, and then protease K is added for incubation to obtain an incubation product B; preferably, the elution buffer used in the elution step comprises 5 mM pH7.5 Tris-HCl, 1 mM EDTA, 0.05% SDS by volume and 1U/. Mu. L RiboLock RNase inhibitor; Preferably, the components of the IP buffer used in the washing step include 10 mM pH 7.5 Tris-HCl, 250mM NaCl, 0.1% NP-40 by volume and 1U/. Mu. L RiboLock RNase inhibitor; preferably, in the eluting step, the addition amount of the proteinase K in the eluting buffer is 1-2% of the total volume of the eluting buffer.
  9. 9. A kit for treating a trace RNA acetylation banking sample is characterized by comprising an antibody magnetic bead incubation reagent, rRNA and DNA removal reagents, an RNA fragmentation reagent, a washing reagent and a concentration reagent; Preferably, the antibody magnetic bead incubation reagent comprises an IP buffer and an anti-ac 4C antibody; Preferably, the rRNA and DNA removal reagents include ：NEBNext v2 rRNA Depletion Solution、NEBNext Probe Hybridization Buffer、NEBNext RNase H Reaction Buffer、NEBNext Thermostable RNase H、Nuclease-free Water、DNase I Reaction Buffer、NEBNext DNase I、Nuclease-free Water、RNAClean XP beads and RiboLock RNase inhibitors; Preferably, the RNA fragmentation reagent comprises RNase inhibitor and IP buffer; preferably, the washing reagent comprises a first concentration buffer solution of RIPA and a second concentration buffer solution of RIPA; Preferably, the elution reagent comprises an elution buffer and proteinase K; preferably, the concentration reagent comprises sodium acetate and linear acrylamide.
  10. 10. Use of the method of any one of claims 1-8 or the kit of claim 9 in sequencing a constructed library.

Description

Micro RNA acetylation library-building sequencing method Technical Field The invention relates to the technical field of biology, in particular to a micro RNA acetylation database-building sequencing method. Background RNA modification is one of the core mechanisms of apparent transcriptome regulation, where N4-acetylcytidine (ac 4C) is currently the only RNA acetylation modification known to exist in both transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA), which is catalyzed by N-acetyltransferase 10 (NAT 10), and plays a key role in RNA stability regulation, translation efficiency regulation and cell physiology. The analysis of apparent transcriptomes is the basis for analyzing the RNA modification function and action mechanism, for example, aiming at high-sensitivity detection technology of RNA modification such as m6A, m5C and the like, the accurate positioning of target modification in a low initial quantity sample is realized, and the functional research of the modification in the fields of cell differentiation, disease occurrence, development and the like is greatly promoted. At present, various detection methods for the detection of ac4C modified whole transcriptome localization have been developed in the prior art, and are mainly classified into three types, antibody-based labeling methods such as acRIP-seq, PA-ac4C-seq and RacRIP-seq, metabolic labeling methods such as FAM-seq, and chemical reaction-based labeling methods such as ac4C-seq. However, the above prior art has the following technical drawbacks, which severely limit the application range of ac4C modification studies. Firstly, the initial RNA consumption is too high, the existing methods (including acRIP-seq, PA-ac4C-seq, racRIP-seq, FAM-seq and ac 4C-seq) generally require a large amount of input RNA samples, for example, the currently widely used acRIP-seq sequencing technology is used for detecting ac4C modification of RNA in cells, the required initial RNA amount reaches 50-500 ug, and the available RNA amount is usually only in nanogram (ng) level in the scenes of rare cell subsets (such as tumor stem cells and specific immune cell subsets) and clinical trace samples (such as puncture biopsy samples), so that the existing methods are not applicable to detection of such samples at all. The detection of some RNA samples with low ac4C modification abundance is greatly limited, and secondly, the detection sensitivity is insufficient, namely, the ac4C modification abundance and stoichiometric amount in mammal mRNA are obviously lower than m6A (usually only one tenth of m 6A), the sensitivity design of the existing method is not specifically optimized for the low ac4C abundance, even under the condition of sufficient RNA usage, the ac4C modification sites with low ac4C abundance are difficult to accurately capture, and the detection requirement of the ac4C modification in the samples with low initial amount cannot be met. In summary, the existing ac4C full transcriptome positioning technology cannot meet the research requirements in the scenes of rare cell subpopulations, trace clinical samples, and RNA sample detection with low ac4C modification abundance due to core defects such as high RNA consumption and low sensitivity. Therefore, there is a need to develop an ac4C modification detection technology with low initial quantity, high sensitivity and wide applicability, so as to fill the technical gap in the field. Disclosure of Invention The invention provides a trace RNA acetylation library construction method, which solves the core defects of high RNA consumption, low sensitivity and the like of the existing ac4C full transcriptome positioning technology by optimizing a impurity washing mode and an rRNA removing mode, and provides a high-efficiency and high-accuracy method for RNA ac4C detection of trace cells and detection of RNA samples with low ac4C modification abundance. In one aspect, the invention provides a method for sequencing a trace RNA in an acetylation database, comprising the following steps: Incubating the magnetic beads with the antibodies to obtain magnetic beads coated with the antibodies; rRNA and DNA removal, namely removing rRNA and DNA in the RNA sample by utilizing RNase H enzyme and DNase I enzyme to obtain a sample with rRNA and DNA removed; the RNA fragmentation step, namely carrying out fragmentation treatment on the sample from which rRNA and DNA are removed to obtain an immune coprecipitation reaction sample; Washing, namely washing the unbound antibodies on the surfaces of the magnetic beads coated with the antibodies, mixing the magnetic beads coated with the antibodies with an immune coprecipitation reaction sample, incubating to obtain an incubation product A, centrifuging the incubation product A to obtain an RNA-antibody-magnetic bead complex, washing the unbound immune coprecipitation reaction sample on the surfaces of the RNA-antibody-magnetic bead complex, and obtaining a washed RN