Improved method for identifying interaction between RNA (Ribonucleic Acid) and RNA binding protein

Abstract

The invention provides an improved method for identifying interaction between RNA and RNA binding protein, and relates to the field of biological analysis. Comprising the following steps: ultraviolet crosslinking: carrying out covalent crosslinking on RNA and binding protein in cells through ultraviolet rays, and fixing instantaneous transition of interaction between RNA and RNA binding protein; s2, cell lysis and fragmentation: splitting the cells treated in the step S1, and then cutting RNA into short fragments by using RNA enzyme; s3, immunoprecipitation: enriching the target RBP and the RNA fragment combined with the target RBP by using a specific antibody; s4, linker connection and library construction: firstly, carrying out reverse transcription on RNA molecules by using a random primer to form a cDNA first chain; then synthesizing a cDNA second chain by using dNTP (deoxyribonucleoside triphosphate) containing dUTP (deoxyuridine triphosphate) and DNA polymerase; carrying out terminal repair and A addition on the double-stranded DNA; connecting a double-chain DNA (deoxyribonucleic acid) joint with a protruding T basic group at the tail end; carrying out PCR amplification to obtain a sequencing library; and S5, performing high-throughput sequencing. The treatment of FastAP and PNK is omitted, the loss of RNA molecules is reduced, and the experimental period is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological analysis, and in particular, to an improved method for identifying RNA and RNA-binding protein interactions. BACKGROUND

[0002] eCLIP-seq (enhanced Cross-Linking and Immunoprecipitation sequencing) is a high-throughput sequencing technology for studying the interaction of RNA-binding proteins (RBPs) with RNA molecules. It is an improvement over traditional CLIP-seq, significantly improving data accuracy and reproducibility by optimizing experimental procedures and reducing background noise. The technology is widely used to reveal RNA metabolism regulation mechanisms, disease-related RNA-protein interaction networks, etc.

[0003] The core principles and steps of traditional CLIP-seq are as follows: UV cross-linking, covalently cross-linking RNA and binding proteins in cells by UV light (254 nm) to fix transient interactions. Cell lysis and fragmentation, after lysing the cells, use RNase to cut the RNA into short fragments (the 3' end of the cut RNA molecule is phosphate, and the 5' end is hydroxyl), leaving the protein binding region. Immunoprecipitation, using specific antibodies to enrich target RBPs and their bound RNA fragments. De-phosphorylation and phosphorylation, using FastAP and PNK reagents to process RNA-protein complexes; linker ligation and library construction, first connecting a specific linker to the 3' end of the RNA fragment, then performing reverse transcription to form RNA-DNA hybrid chains. Then connect a specific linker to the 3' end of the DNA fragment. Primer matching specific linker for PCR amplification to obtain sequencing library. High-throughput sequencing, sequence analysis of binding sites by next-generation sequencing (such as Illumina).

[0004] Key improvements of eCLIP over traditional CLIP: introduction of dual-end unique molecular identifiers (UMIs) to reduce PCR amplification bias. Optimization of RNA library construction method to retain RNA molecules that terminate reverse transcription at the UV cross-linking site. Standardization of control experiments (such as Size-matched Input control group) to improve data analysis reliability.

[0005] However, the eCLIP technology has the following defects: the specific linker at the end needs to ensure that the 3' end of the RNA fragment is hydroxyl and the 5' end is phosphate, which requires phosphorylation and de-phosphorylation treatment, resulting in a certain amount of RNA fragment loss. After reverse transcription, a specific linker is connected to the 3' end of the DNA fragment, and the connection efficiency between such single-stranded DNA molecules is very low, requiring an overnight reaction with a long time period. SUMMARY

[0006] The application aims to provide an improved method for identifying RNA and RNA-binding protein interactions, which omits the processing of FastAP and PNK, reduces the loss of RNA molecules, and shortens the experimental period.

[0007] The application solves the technical problem by using the following technical scheme.

[0008] The application provides an improved method for identifying RNA and RNA-binding protein interactions, comprising the following steps:

[0009] S1, UV crosslinking: covalently crosslinking the RNA and the binding protein in the cell by ultraviolet light, fixing the transient state of the RNA and the RNA-binding protein interaction;

[0010] S2, cell lysis and fragmentation: lysing the cell treated in step S1, and then cutting the RNA into short fragments by using RNase, wherein the 3' end of the RNA molecule is phosphate and the 5' end is hydroxyl, and the protein binding region is reserved;

[0011] S3, immunoprecipitation: using specific antibodies to enrich the target RBP and the RNA fragments bound thereto;

[0012] S4, linker connection and library construction: first, reverse transcribing the RNA molecule into a cDNA first strand by using a random primer; then, synthesizing a cDNA second strand by using dNTP containing dUTP and a DNA polymerase; performing end repair and A addition on the double-stranded DNA; connecting the double-stranded DNA linker with the protruding T base; and performing PCR amplification to obtain a sequencing library;

[0013] S5, high-throughput sequencing: analyzing the binding site sequence by using second-generation sequencing.

[0014] In some embodiments of the application, in the step S1, the covalently crosslinking the RNA and the binding protein in the cell by ultraviolet light comprises:

[0015] Under ice bath, the cell is treated by using a UV crosslinking instrument, and after natural decay to zero, the cell is centrifuged at 4 DEG C for 5 min, the supernatant is removed, the cell is rinsed with 1 mL of pre-cooled 1* PBS for 1-2 times, and then the cell is stored at-80 DEG C.

[0016] In some embodiments of the application, in the step S2, the lysis of the cell comprises:

[0017] The lysis solution, recombinant RNase inhibitor, and protease inhibitor are added into the cell, and then mixed uniformly, and the cell is incubated at 4 DEG C for 30 min, and then ultrasonically treated for 5 min.

[0018] In some embodiments of the present application, after lysing the cells, DNA is also digested:

[0019] To the lysate after ultrasonic treatment, 10x RQI buffer is added, mixed uniformly at 37°C, and digested for 30 min.

[0020] In some embodiments of the present application, in the step S3, the specific antibody is IGF2BP2 and negative control IgG antibody.

[0021] In some embodiments of the present application, in the step S3, the immunoprecipitation comprises:

[0022] Antibody-magnetic bead incubation, lysate-antibody-magnetic bead incubation, NuPAGE gel electrophoresis separation of protein-RNA complex.

[0023] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0024] The method for identifying the interaction between RNA and RNA binding protein provided by the present application saves the treatment of FastAP and PNK, reduces the loss of RNA molecules, and shortens the experimental period. The RNA molecules are reverse transcribed to synthesize the first and second strands of cDNA, and then the double-stranded DNA specific adapter is connected through the complementary pairing of the terminal A-T base, the connection efficiency is greatly improved, and the library construction success rate is improved. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0026] Figure 1 For IP efficiency detection of IGF2BP2 antibody of the embodiments of the present application;

[0027] Figure 2 For agarose gel electrophoresis of the sequencing library constructed in the embodiments of the present application;

[0028] Figure 3 For fragment size distribution graph of the sequencing library in the embodiments of the present application;

[0029] Figure 4 For the distribution proportion of peak in different regions of the reference genome in the embodiments of the present application;

[0030] Figure 5The motif analysis result of the peak of the embodiment of the present application. DETAILED DESCRIPTION

[0031] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, conventional conditions or conditions suggested by manufacturers are adopted. If the manufacturers of reagents or instruments are not indicated, all are conventional products that can be obtained by market purchase.

[0032] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to specific embodiments.

[0033] EMBODIMENT

[0034] (1) UV crosslinking cells:

[0035] Ice cubes were laid in a tray, and a culture dish was placed on the ice cubes. The tray was placed in a UV crosslinking instrument, and 400 mj / cm2(254 nm) was set. Natural decay was performed to zero, 4℃, centrifugal force was 200xg, and centrifugation was performed for 5 min. Supernatant was removed. 1 mL of pre-cooled 1xPBS was used to rinse the cells for 1-2 times, and the cells were stored at -80℃. Among them, 1xPBS rinsing the cells means rinsing the cells with 1-fold concentration of PBS.

[0036] (2) Lysis of cells:

[0037] About 100 μL of dry volume of cells were taken, and lysis buffer was added at a ratio of 1:10, RNase enzyme inhibitor (RRI, TakaRa) was added at a ratio of 1%, and protease inhibitor (cocktail) was added at a ratio of 1%. After mixing, 4℃ rotation incubation was performed for 30 min, and the rotation speed was set to 10.

[0038] The composition of the lysis buffer Lysis buffer: 50 mM Tris-HCl, pH=7.4, 100 mM NaCl, 1% octylphenol polyoxyethylene ether (NP-40), 0.1% sodium dodecyl sulfate (SDS), and 0.5% sodium deoxycholate.

[0039] (3) Ultrasonic lysis of the lysis buffer:

[0040] A Ningbo Xinzhi ultrasonic cell pulverizer (JY92-IIN) was used, 10% power (65 W) was set, and pulverization was performed for 10 s, pause for 10 s, and then ultrasonic was performed for 5 min.

[0041] (4) Digestion of DNA:

[0042] Take 900 μL of the lysate after sonication, add 100 μL of 10x RQ I buffer, 50 μL of DNase I (RQ I), and run the thermomixer at 37°C, 1,200 rpm, 20 s on, 3 min off, for 30 min. Take 20 μL of the lysate after sonication and 20 μL of the lysate after DNase I (RQ I) digestion, add 2 μL of 10 mg / mL proteinase K, and run the thermomixer at 50°C, 1,200 rpm, 20 s on, 3 min off, for 20 min. Add 22 μL of phenol:chloroform:isoamyl alcohol (pH > 8.0), shake the mixture, centrifuge at 13,200 rpm for 15 min at 4°C, and take 15 μL of the supernatant. Add loading buffer and run 1.5% agarose gel electrophoresis to detect whether the DNA is digested completely.

[0043] (5) RNA quantification and enzymatic fragmentation:

[0044] Take 20 μL of the lysate after DNase I (RQ I) digestion, add 2 μL of 10 mg / mL proteinase K, and run the thermomixer at 50°C, 1,200 rpm, 20 s on, 3 min off, for 20 min. Use the RNA Clean & Concentrator™-5 (Zymo research, R1015) kit to purify the RNA. Add 78 μL of DEPC-H2O to a total volume of 100 μL. Add 200 μL of RNA binding buffer (RNA Binding Buffer) and mix well. Add 300 μL of ethanol and mix well. Transfer the entire solution to a Zymo-Spin™-IC Column, centrifuge at 10,000 xg (10,000 times the acceleration due to gravity) for 30 s, and transfer the centrifuged solution to the Zymo-Spin™-IC Column again. TM Centrifuge at 10,000 xg for 30 s. Discard the supernatant, add 400 μL of RNA preparation buffer (RNA Prep Buffer), and centrifuge at 10,000 xg for 30 s. Discard the supernatant, add 700 μL of RNA wash buffer (RNA Wash Buffer), and centrifuge at 10,000 xg for 30 s. Discard the supernatant, add 400 μL of RNA wash buffer (RNA Wash Buffer), and centrifuge at 10,000 xg for 1 min. Discard the supernatant, open the cap, and place it at room temperature for 5 min. Add 10 μL of DEPC-H2O, place it at room temperature for 1 min, centrifuge at 10,000 xg for 1 min, and add 10 μL of DEPC-H2O again. Place it at room temperature for 1 min, centrifuge at 10,000 xg for 1 min, and take 1 μL for quantification using Qubit. Reserve the remaining RNA sample as an RNase I fragmentation control sample. TM Centrifuge at 10,000 xg for 30 s. Discard the supernatant, add 400 μL of RNA preparation buffer (RNA Prep Buffer), and centrifuge at 10,000 xg for 30 s. Discard the supernatant, add 700 μL of RNA wash buffer (RNA Wash Buffer), and centrifuge at 10,000 xg for 30 s. Discard the supernatant, add 400 μL of RNA wash buffer (RNA Wash Buffer), and centrifuge at 10,000 xg for 1 min. Discard the supernatant, open the cap, and place it at room temperature for 5 min. Add 10 μL of DEPC-H2O, place it at room temperature for 1 min, centrifuge at 10,000 xg for 1 min, and add 10 μL of DEPC-H2O again. Place it at room temperature for 1 min, centrifuge at 10,000 xg for 1 min, and take 1 μL for quantification using Qubit. Reserve the remaining RNA sample as an RNase I fragmentation control sample.

[0045] Take total amount 100 mg RNA corresponding to the lysis solution after ultrasonic, add 40 U RNase I, hot mixer 37℃, 1,200 rpm, 20 s on, 3 min off, digestion 5 min. Take 20 μL RNase I digestion after lysis solution, add 2 μL 10 mg / mL proteinase K, hot mixer 50℃, 1000 rpm, 20 s on, 3 min off, digestion 20 min. Add 22 μL phenol: chloroform: isopropyl (pH <5.0) shake mixed sample, 4℃, 13200 rpm, centrifugation 15 min. Take 15 μL supernatant, add loading buffer, run 1.5% agarose gel electrophoresis to detect RNA fragment size. The remaining RNA sample in the previous step is used as a RNase I fragmentation control sample for electrophoresis detection.

[0046] (6) Immunoprecipitation:

[0047] Antibody-magnetic bead incubation: take 50 μL protein A / G magnetic beads, place in magnetic field for 3 min, discard supernatant. 1 mL pre-cooled lysis buffer is used to rinse the magnetic beads twice. Resuspend the magnetic beads with 100 μL lysis buffer, add 10 μg IGF2BP2 and negative control IgG antibody, 4℃, 10 rpm, rotate incubation for 120 min.

[0048] Lysis buffer-antibody-magnetic bead incubation: add 100 mg RNA corresponding lysis buffer product after RNase I digestion, resuspend the magnetic beads, 4℃, 10 rpm, rotate incubation overnight. After antibody-magnetic bead incubation, take 2% volume of solution (containing magnetic beads) as SMInput. Place in magnetic field for 3 min, retain the supernatant. Place in magnetic field, discard the supernatant. 1 mL pre-cooled high-salt buffer is used to rinse the magnetic beads twice. Place in magnetic field for 3 min, discard the supernatant. 1 mL pre-cooled wash buffer is used to rinse the magnetic beads twice. Take 1 / 5 volume of magnetic beads, resuspend with 20 μL elution buffer, at the same time take 10-20 μL WB-Input sample, add 5 times volume of SDS loading buffer, 99℃, 1200 rpm, incubate for 10-15 min.

[0049] Wash buffer (Wash buffer) components: 20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% (vol / vol) Tween-20 and 5 mM NaCl.

[0050] High-salt wash buffer components: 50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate.

[0051] (7) NuPAGE gel electrophoresis separation of protein-RNA complex:

[0052] According to the protein molecular weight size of the study, select 12% or 4-12% NuPAGE gel. Dilute 20X MOPS buffer to 1X. Pre-electrophoresis at 150V for 10 min. Leave a gel hole between each sample, add 20 μL of 1X SDS loading buffer. Protein Mark points in the leftmost gel hole. Electrophoresis at 150V for 75 min or until the target protein is separated. Cut the gel block in the target protein molecular weight size +75 kDa region, put it into a bottom-punched 500 μL EP tube, and then into a 1.5 mL EP tube. 4°C, 13200 rpm, centrifugation for 3 min.

[0053] (8) Extraction of RNA:

[0054] Add 400 μL of lysis solution, 40 μL of proteinase K solution, 50°C, 1200 rpm, incubate for 120 min. Add an equal volume of phenol: chloroform: isopropyl alcohol (pH <5.0), 4°C, 12000 rpm, centrifuge for 3 min. Use the RNA Clean & Concentrator™-5 (Zymo research, R1015) kit to purify the RNA. Add 800 μL of RNA binding buffer and mix well. Add 1200 μL of ethanol and mix well. Transfer the entire solution to a Zymo-Spin™ IC Column. 10000xg, centrifuge for 30 s. Transfer the centrifuged solution to a Zymo-Spin™ IC Column again. TM IC Column, 10000xg, centrifuge for 30 s. Transfer the centrifuged solution to a Zymo-Spin™ IC Column again. TMIn the IC Column, 10000xg, centrifuge for 30s. Discard the supernatant, add 400 μL RNA preparation buffer (RNA Prep Buffer), 10000xg, centrifuge for 30s. Discard the supernatant, add 700 μL RNA rinse buffer (RNA Wash Buffer), 10000xg, centrifuge for 30s. Discard the supernatant, add 400 μL RNA rinse buffer (RNA Wash Buffer), 10000xg, centrifuge for 1 min. Discard the supernatant, open the tube cap, and place it at room temperature for 5 min. Add 6 μL DEPC-H2O, place it at room temperature for 1 min, 10000xg, centrifuge for 1 min. Add 6 μL DEPC-H2O again, place it at room temperature for 1 min, 10000xg, centrifuge for 1 min.

[0055] (9) RNA sample library construction:

[0056] The Input and IP RNA samples were subjected to sequencing library construction using the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina (Vazyme, NR606-01) kit.

[0057] 1. Preparation of reaction system:

[0058] Component Volume RNA 8 μL 2x Frag / Prime Buffer V2 8 μL

[0059] Gently pipette 10 times to mix thoroughly. Place the sample in a PCR machine at 70°C for 3 min; immediately place it on ice for 3 min.

[0060] 2. Double-stranded cDNA synthesis

[0061] Take the components required for double-stranded cDNA synthesis from -30 to -15°C, thaw on ice, mix well by inverting, and collect at the bottom of the tube by brief centrifugation. Prepare the first-strand cDNA synthesis reaction system according to the following table:

[0062] Component Volume Product from previous step 16 μL 1st Strand Buffer 6 7 μL 1st Strand Enzyme Mix 4 2 μL

[0063] Adjust the pipette to 20 μl scale, and gently pipette 10 times to mix thoroughly. Perform the first-strand cDNA synthesis reaction in a PCR machine:

[0064] Temperature Time Hot lid 105 °C ON 25℃ 10 min 42℃ 15 min 70℃ 15 min 4℃ Hold

[0065] Prepare the second-strand cDNA synthesis reaction system according to the following table:

[0066] Component Volume 1st Strand cDNA from previous step 25 μL 2nd Strand Buffer 3 (with dNTP or dUTP) 25 μL 2nd Strand Enzyme Super Mix 3 15 μL

[0067] Adjust the pipette to the 50 μl range and pipette up and down 10 times to mix well. Perform the second strand cDNA synthesis reaction in a PCR machine:

[0068] Temperature Time Hot lid 105 °C ON 16℃ 30 min 65℃ 15 min 4℃ Hold

[0069] The double strand synthesis product can be stored at -30 to -15 °C for 24 h.

[0070] 3. Ligation

[0071] Prepare the ligation system as follows according to the table below:

[0072] Component Volume ds cDNA from previous step 65 μl Rapid Ligation Buffer 6 25 μl Rapid DNA Ligase 6 5 μl Adapter 5 μl

[0073] Adjust the pipette to the 80 μl range and pipette up and down 10 times to mix well.

[0074] Perform the ligation reaction in a PCR machine:

[0075] Temperature Time Hot lid 105 °C ON 20℃ 15 min 4℃ Hold

[0076] 4. Product purification

[0077] Take the VAHTS DNA Clean Beads out of the 2-8 °C 30 min in advance and let it equilibrate to room temperature. Mix the VAHTS DNA Clean Beads well by inverting or vortexing, pipette 120 μl (1.2x) into the ligation product and mix well by pipetting up and down 10 times gently. Incubate at room temperature for 10 min to bind the DNA to the magnetic beads. Place the sample on a magnetic stand and remove the supernatant carefully when the solution is clear (about 5 min). Keep the sample on the magnetic stand and add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads (be careful not to blow the magnetic beads away), incubate at room temperature for 30 sec, and remove the supernatant carefully. Repeat the previous step once. Keep the sample on the magnetic stand and dry the magnetic beads at room temperature for about 5 min with the lid open. Take the sample off the magnetic stand, add 22 μl of Nuclease-free ddH2O, mix well by pipetting up and down, and place it on the magnetic stand after incubating at room temperature for 2 min. When the solution is clear (about 5 min), carefully pipette 20 μl of the supernatant into a new Nuclease-free PCR tube.

[0078] 5. PCR amplification

[0079] Prepare the PCR reaction system according to the table below according to the use of the adapter:

[0080] Component Volume Purified Adapter Ligation Product 20 μl i5 Primer 2.5 μl i7 Primer 2.5 μl VAHTS HiFi Amplification Mix 3 25 μl

[0081] Adjust the pipette to the 30 μl range and pipette up and down 10 times to mix well. Place the sample in the PCR machine and run the library amplification reaction:

[0082] Step Temperature Time Cycle Number Hot lid 105℃ ON Pre-denaturation 98℃ 45 sec 1 Denaturation 98℃ 15 sec 15 Annealing 60℃ 30 sec 15 Extension 72℃ 30 sec 15 Full extension 72℃ 1 min 1 4℃ Hold 1

[0083] 6. Product agarose gel excision and recovery

[0084] Prepare 2.5% agarose gel and run the electrophoresis. Run the electrophoresis for 60 min at gradient voltage. Excise the band between 150-300 bp under the UV light. Use Qiange gel recovery kit to recover the DNA. Add 6 times volume of Buffer QG according to the weight of the gel piece (100 mg weight corresponds to 100 μL) and incubate at 50 °C for 10 min. Add equal volume of isopropanol and mix well according to the weight of the gel piece (100 mg weight corresponds to 100 μL). Centrifuge at 13000 rpm for 1 min and discard the supernatant. Add 500 μL Buffer QG, centrifuge at 13000 rpm for 1 min and discard the supernatant. Add 750 μL Buffer PE, centrifuge at 13000 rpm for 1 min and discard the supernatant. Centrifuge at 13000 rpm for 1 min, open the tube cap and dry at room temperature for 5 min. Add 10 μL Buffer EB, place at room temperature for 1 min and centrifuge at 13000 rpm for 1 min.

[0085] (10) Library sequencing

[0086] Run PE150 sequencing on illumina sequencing instrument and the results are shown in the attached figures.

[0087] Figure 1 To use IGF2BP2 antibody to immunoprecipitate and enrich IGF2BP protein and its bound RNA. The antibody immunoprecipitation enrichment was detected by WB experiment, and very obvious IGF2BP2 protein band was detected in the IP sample, indicating that the IGF2BP2 antibody immunoprecipitation experiment was successful.

[0088] Using the extracted RNA sample, use the kit to build a library. Figure 2The figure is the result of 2.5% agarose gel electrophoresis of the product after 12 cycles of PCR amplification. The 1-2 wells are two input samples, and the 3-4 wells are two IGF2BP3 antibody IP samples. The IGF2BP3 antibody IP sample PCR product has a brighter band at about 150 bp than the input sample, indicating that the IGF2BP3 antibody IP sample has more small fragment RNA, which is consistent with the characteristics of CLIP-seq technology, that is, more small fragment RNA is enriched, which can more accurately predict the RNA characteristics of RNA-binding protein binding. The 150-300 bp PCR product was recovered by gel cutting and used as a library for PE150 sequencing on an illumina sequencing instrument.

[0089] Figure 3 The length distribution of the sequencing reads is the length size distribution result of the IGF2BP2 antibody IP sample reads. It can be seen that there are small fragment reads with a size of 30-130 bp. Analysis of these small fragment reads can more accurately analyze the location information of the RNA bound by the RNA-binding protein on the gene, and accurately analyze and predict the sequence characteristics (motif base sequence) of the bound RNA.

[0090] Figure 4 The figure is the result of using ABLIRC software to analyze the IGF2BP2 antibody enrichment sample relative to the input sample on the gene binding peak (peak). This figure is a statistical analysis of the proportion of peaks in different regions of the genome. The binding peak (peak) is enriched in the CDS and three prime UTR regions, which is consistent with the function of IGF2BP2 as an RNA-m6A modification reading protein.

[0091] Figure 5 The figure is the result of using HOMER software to analyze the motif of these peak sequences, which is the top 5 result of IGF2BP2 antibody enrichment sample Homer de novo Motif.

[0092] In summary, the method for identifying RNA and RNA-binding protein interactions provided by the present application eliminates the FastAP and PNK processing, reduces the loss of RNA molecules, and shortens the experimental period. The RNA molecules are reverse transcribed to synthesize the first and second strands of cDNA, and then the double-stranded DNA specific adapter is connected through the complementary pairing of the terminal A-T base, the connection efficiency is greatly improved, and the library construction success rate is improved.

[0093] The embodiments described above are only part of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only to represent selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

Claims

1. An improved method for identifying the interaction between RNA and RNA-binding proteins, characterized in that The following steps are involved: S1, UV crosslinking: UV light is used to covalently crosslink RNA and binding proteins in cells, fixing the transient state of interaction between RNA and RNA-binding proteins; S2, cell lysis and fragmentation: Lyse the cells treated in step S1, and then use RNase to cut the RNA into short fragments. After cutting, the 3' end of the RNA molecule is phosphate and the 5' end is hydroxyl, retaining the protein binding region; S3, immunoprecipitation: using specific antibodies to enrich the target RBP and its bound RNA fragments; S4, adapter ligation and library construction: First, reverse transcribe the RNA molecule with random primers to form the first strand of cDNA; then synthesize the second strand of cDNA using dNTPs containing dUTP and DNA polymerase; end-repair and A addition of double-stranded DNA; ligate double-stranded DNA adapters with overhanging T bases at the end; PCR amplification to obtain sequencing library; S5, high-throughput sequencing: analysis of binding site sequences by next-generation sequencing.

2. The improved method for identifying the interaction between RNA and RNA-binding protein according to claim 1, characterized in that In step S1, the covalent cross-linking of the intracellular RNA and the binding protein by ultraviolet light comprises: In an ice bath, use a UV cross-linker to treat the cells. After natural decay to zero, centrifuge at 4°C for 5 minutes, remove the supernatant, add 1 mL of pre-cooled 1× PBS to rinse the cells 1-2 times, and then freeze at -80°C.

3. The improved method for identifying the interaction between RNA and RNA-binding protein according to claim 1, characterized in that In step S2, the cell lysis comprises: Lysis buffer, recombinant RNase inhibitor, and protease inhibitor were added to the cells, mixed well, incubated with rotation at 4°C for 30 min, and then sonicated for 5 min.

4. The improved method for identifying the interaction between RNA and RNA-binding protein according to claim 3, characterized in that: After cell lysis, DNA is also digested: RQI buffer was added to the lysate after ultrasonic treatment, mixed evenly, and digested at 37°C for 30 min.

5. The improved method for identifying the interaction between RNA and RNA-binding protein according to claim 1, characterized in that In step S3, the specific antibodies are IGF2BP2 and negative control IgG antibodies.

6. The improved method for identifying the interaction between RNA and RNA-binding protein according to claim 1, characterized in that In step S3, the immunoprecipitation comprises: Antibody-magnetic bead incubation, lysate-antibody-magnetic bead incubation, NuPAGE gel electrophoresis separation of protein-RNA complexes.

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