CN111560423B - Method for detecting RNA m6A with high flux and high sensitivity and single base resolution and application thereof - Google Patents
Method for detecting RNA m6A with high flux and high sensitivity and single base resolution and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting RNA m6A with high flux, high sensitivity and single base resolution and application thereof. The method comprises the following steps: (1) Extracting total RNA in a sample and removing rRNA to obtain an RNA sample; (2) Fragmenting the RNA sample into 2 parts, namely RNA fragments I and II; (3) subjecting the RNA fragment I to co-immunoprecipitation and crosslinking treatment; (4) Respectively carrying out 3' -terminal joint connection on the complex obtained by crosslinking treatment and the RNA fragment II; (5) Respectively carrying out reverse transcription on the cDNA and connecting the cDNA with a 3' -terminal connector, and carrying out PCR amplification to respectively construct an hmCLIP library and an input library; (6) The hmCLIP library and the input library were sequenced using a high throughput sequencing platform. The method provided by the invention has the advantages of short time consumption and high sensitivity, and is suitable for detecting RNA m6A in a small amount of samples and samples with low RNA integrity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting RNA m6A with high flux and high sensitivity and single base resolution and application thereof.
Background
The m6A modification of RNA occurs at the N6 position of the adenylate of RNA, is the most common type of apparent post-transcriptional modification on eukaryotic mRNA, and accounts for about 0.1-0.4% of adenine on mRNA. The m6A modification of RNA is produced by the catalysis of the methyltransferase complex and demethylase, and is therefore a dynamic reversible process. More and more studies indicate that m6A plays an important role in important life processes such as embryonic development, neurogenesis, tumor progression, sex determination, etc. Thus, accurate localization of the m6A modification site of RNA is extremely important for studying the specific mechanism of action of m6A in regulating vital activities. However, m6A has similar physicochemical properties to A, does not affect the ability of the base to pair, and there has not been a chemical agent capable of altering this modification.
With the development of antibody-dependent second generation sequencing technologies meRIP and migip, important biological functions of m6A are gradually revealed. meRIP fragments RNA first, then in two parts: one part is enriched by an anti-m 6A antibody, then the enriched RNA fragments are subjected to library construction to be used as a meRIP group, the other part is used for common RNA library construction to be used as an input group, and the m6A modified region is determined by analysis through a bioinformatics means; the principle of the MICLIP is similar to that of the meRIP, but different from that of the meRIP, the m6A antibody is firstly used for enriching fragmented RNA, the m6A antibody-RNA complex obtained through enrichment is subjected to ultraviolet 254nm crosslinking, an amino acid residue is remained at a crosslinking site according to the characteristic that the crosslinking product is digested by proteinase K, C-T mutation can occur in the reverse transcription process, and the m6A modification site can be distinguished by a single base by utilizing a bioinformatics means. Although both meRIP and migip enable m6A information detection at the full transcriptome level, there are some drawbacks: 1. the two methods have large demand on sample RNA, the dosage of the RNA is about 500ug, and m6A detection is difficult to realize in rare samples; 2. the meRIP is realized by a method for enriching the RNA according to the m6A antibody after fragmenting, and finally the fragment enriched by the antibody and provided with the m6A site is obtained, so that the resolution is lower; 3. the MICLIP is only used for determining mutation sites through peaks obtained by antibody enrichment, and has higher false positive rate for RNA with higher background expression level; 4. the flow of the MICLIP is complex, the steps are more, and the requirement on experimental conditions is higher. In order to better study the function and the action mechanism of the m6A, the establishment of an efficient, sensitive and single-base resolution m6A detection method is significant.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a method for detecting RNA m6A with high throughput and high sensitivity and single base resolution.
It is another object of the present invention to provide the use of the method for high throughput high sensitivity single base resolution detection of RNA m 6A.
The aim of the invention is achieved by the following technical scheme:
a method for detecting RNA m6A with high flux and high sensitivity single base resolution, comprising the following steps:
(1) Preparation of RNA samples for DerRNA
Extracting total RNA in a sample, and then removing rRNA in the total RNA to obtain an RNA sample;
(2) RNA fragmentation
Fragmenting the RNA sample obtained in the step (1) to obtain RNA fragments, dividing the RNA fragments into 2 parts, and naming the RNA fragments as RNA fragments I and II;
(3) Co-immunoprecipitation and cross-linking
Performing heat denaturation treatment on the RNA fragment I obtained in the step (2), and then reacting with an antibody specifically recognizing m6A locus to obtain a reaction solution; then, the reaction solution is crosslinked under the ultraviolet condition (covalent crosslinking sites are formed between the recognition sites and the antibody) to obtain a crosslinked complex; adding protein A/G beads into the cross-linked complex for co-immunoprecipitation to obtain an co-immunoprecipitation product; wherein, the conditions of crosslinking are: 150-1500 mj/cm 2 UV crosslinking at 254nm for 2-4 times;
(4) RNA adaptor ligation
(1) Connecting the co-immunoprecipitation product obtained in the step (3) with a fluorescent labeling joint at the 3' -end of RNA, washing off redundant joints after connection, carrying out denaturation elution, and carrying out electrophoresis separation to obtain a crosslinked connection product; then adding proteinase K into the cross-linked connection product for digestion, and recovering RNA by using at least one of silane magnetic beads, naCl or PEG 8000 after digestion to obtain purified RNA;
(2) connecting the RNA3' terminal joint of the RNA fragment II obtained in the step (2) and recovering to obtain RNA connected by the joint;
(5) Building a warehouse
Reverse transcription of the purified RNA obtained in step (4) (1) into cDNA, and then 3' -terminal adaptor ligation of the cDNA to obtain adaptor-ligated cDNA; carrying out PCR amplification and purification (fragment size screening) on the cDNA connected with the connector to obtain a PCR product, namely constructing an hmCLIP library (m 6A-CLIP library); replacing the RNA after the linker connection obtained in the step (4) (2) with the purified RNA obtained in the step (4) (1), and constructing an input library according to the same operation steps;
(6) Sequencing
Sequencing the hmCLIP library and the input library constructed in the step (5) by using a high-throughput sequencing platform.
The sample in the step (1) is a paraffin specimen of a cell, a tissue or a pathological section; preferably paraffin samples; more preferably a thyroid cancer fine needle puncture paraffin specimen.
The total RNA extraction described in step (1) may be performed using methods conventional in the art or RNA extraction kits; extraction is preferably performed using a QIAGEN RNeasy FFPE kit kit.
The rRNA in the total RNA removed in the step (1) can be removed by a method or a kit conventional in the art, and other types of RNA (such as circular RNA, non-polyA tail noncoding RNAs) except RNA with polyA tail can be reserved; preferably using Ribo-off TM rRNA depletion kit (Human/Mouse/Rat) (Northenzan) kit removes rRNA.
The mass of the RNA fragment I in the step (2) is more than or equal to 100ng; preferably not less than 200ng.
The mass of the RNA fragment II in the step (2) is more than or equal to 10ng; preferably not less than 50ng.
The thermal denaturation treatment conditions described in the step (3) are: treating at 75 deg.c for 5min and then setting on ice for 2-3 min.
The antibody specifically recognizing the m6A site in the step (3) is an m6A antibody (m 6A antibody); preferably, the m6A antibody is purchased from Abcam corporation.
The conditions for crosslinking described in step (3) are preferably: 150-400 mj/cm 2 UV crosslinking at 254nm for 2-4 times; more preferably: 150mj/cm 2 UV crosslinking at 254nm was performed 4 times.
The conditions for co-immunoprecipitation described in step (3) are preferably: incubate at 4℃for 2h.
The electrophoresis separation in the step (4) (1) is carried out by adopting 4% -12% of NuPAGE.
The dosage of NaCl in the step (4) (1) is calculated according to the addition of 45-55 nmol/L of the final concentration of the NaCl in the reaction system; preferably, the addition is carried out at a final concentration of 50nmol/L in the reaction system.
The PEG8000 in step (4) (1) is preferably a 50% strength by volume PEG8000 solution (i.e., 100ml water containing 50g of PEG 8000).
The dosage of the PEG8000 in the step (4) (1) is calculated according to the addition of the PEG8000 with the final concentration of 0.04-0.06 g/ml in the reaction system (namely, the addition of the PEG8000 with the final concentration of 4-6% (w/v)); preferably, the catalyst is added in such a manner that the final concentration of the catalyst in the reaction system is 0.05g/ml by mass (i.e., the catalyst is added in a final concentration of 5% (w/v)).
In the electrophoresis described in the step (4) (1), the uncrosslinked complex (i.e., the reaction solution obtained in the step (3)) may be used as a control for distinguishing false positives caused by incomplete denaturation.
The T4 RNA Ligase used for the adaptor ligation described in step (4) was T4 RNA Ligase 1,High Concentration.
The dosage of the T4 RNA ligase is calculated according to the addition of 2000U/ml of the final concentration of the T4 RNA ligase in the reaction system
The 3' -end fluorescent labeling joint of the RNA in the step (4) adopts an end azide modification group, and reacts with an infrared dye with DBCO label by adopting a click chemistry principle, so that the RNA joint can be visually identified when being irradiated by infrared rays, and the sequence is as follows:
5 Phos/AUAUAGGNNNAGNNAUCGGAAGCGUCGUGUAG/3 Azidec/(N: a, c, g, t oru; phos: phosphate group modification; azide: azide modification).
The denaturation and elution in the step (4) (1) are realized by the following steps: co-immunoprecipitation of products (antibody RNA complexes)Sample Reducing Agent (Thermo, cat# NP 0009) and denaturing elution to distinguish uncrosslinked from crosslinked samples, the position of the crosslinked product can be determined.
The silane beads described in step (4) (1) were Dynabeads MyOne Silane (Thermo, cat# 37002D) beads.
The addition of random bases (NNNNN) to the 5' -end of the RNA adapter described in step (4) allows discrimination between false positives resulting from subsequent post-reverse transcription PCR amplification.
The primer sequences required for reverse transcription described in step (5) are as follows:
5’-ACACGACGCTCTTCCGA-3’。
The purification described in step (5) was performed using Agencourt AMpure XP beads.
The 3' -terminal linker sequence described in step (5) is as follows:
5Phos/NNNNNNNNNNAGATCGGAAGAGCACACGTCTG/3 SpC/(N: a, c, g, t or u; phos: phosphate group modification; spC3: spacers modification C3 spacer, preventing self-ligation of the linker).
The high throughput sequencing platform described in step (6) is preferably a high throughput sequencing using the Hiseq X10 platform from illuminea.
The method for detecting RNA m6A with high flux and high sensitivity single base resolution is applied to the detection of RNA m 6A.
The application field is the non-disease diagnosis and treatment field.
The detected sample comprises a cell, a tissue or a pathological section paraffin specimen; preferably paraffin samples; more preferably a thyroid cancer fine needle puncture paraffin specimen.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention establishes a method for detecting m6A locus by full transcriptome level high sensitivity single base, reduces the RNA consumption to about 2ug, and can be suitable for a small amount of samples (rare samples) and samples with low RNA integrity, such as paraffin samples, and compared with the existing 500ug RNA, the sensitivity is greatly improved.
(2) The crosslinking condition of the antibody-RNA complex is further optimized, so that the yield of a crosslinked product is optimal, and the antibody enrichment efficiency is obviously improved;
(3) According to the invention, input is introduced for the first time on the basis of single base detection m6A, so that the false positive rate of CLIP is reduced.
(4) The RNA purification step in the proteinase K digestion product of the m6A antibody-RNA utilizes silane silica gel magnetic beads to separate and purify the RNA, and further improves the recovery rate by about 5 times through improving the buffer (buffer) component, and compared with the existing ethanol precipitation method and zymo column extraction method, the method has the advantages of short time consumption and high recovery efficiency.
(5) The invention realizes the full transcriptome level detection of RNA m6A locus in a tissue degradation specimen (paraffin specimen) for the first time.
(6) The invention can realize the identification of the site of the full transcriptome level m6A in a small amount of samples including a small amount of RNA degradation samples, and lays a foundation for further researching the function of m6A in rare samples.
Drawings
Fig. 1 is a schematic flow chart of the hmCLIP of the present invention.
FIG. 2 is a graph showing the results of the detection and analysis of a library Agilent bioanalyzer 2100 obtained after library construction; wherein a is the Agilent bioanalyzer 2100 result of the input library; b is Agilent bioanalyzer 2100 results for the hmCLIP library.
FIG. 3 is a graph of m6A conserved motif and distribution characteristics in a paraffin specimen using the method of the present invention; wherein A is m6A conservative motif; b is m6A distribution characteristics.
FIG. 4 is a single base resolution of the overlap of the m6A position with the position where the CLIP causes a C.fwdarw.T mutation or termination; wherein A is the overlapping degree of single base resolution m6A position and C-T mutation position caused by CLIP; b is the overlap of the single base resolution m6A position with the position where the CLIP caused termination.
FIG. 5 is an example of methylation patterns of individual genes, as compared to existing m6A-CLIP data.
FIG. 6 is a graph of m6A enrichment of different regions of a single gene verified by meriP-qPCR.
FIG. 7 is a graph showing the effect of different crosslinking conditions on crosslinking effect (including UV 254nm 150mJ/cm 2 *2：UV 150mJ/cm 2 Crosslinking for 2 times; 150mJ/cm 2 *4：UV 150mJ/cm 2 Crosslinking for 4 times; 300mJ/cm 2 *2：300mJ/cm 2 Crosslinking for 2 times; 400mJ/cm 2 *2：400mJ/cm 2 Crosslinking for 2 times; 1500mJ/cm 2 *2：1500mJ/cm 2 Crosslinking 2 times); wherein A represents the imaging result of the adelsteine machine; b is the quantized value of A.
FIG. 8 is a graph of the results of a reproducibility test of the efficiency of ligation of high concentration ligase to common ligase; wherein A represents the imaging result of the adelsteine machine; b is the quantized value of A.
FIG. 9 is a graph showing the results of optimization of RNA silane bead purification (NaCl addition, PEG 8000) in proteinase K digestion products of m6A antibody-RNA complexes and comparison with the conventional ethanol precipitation method and zymo column method; wherein A represents the imaging result of the adelsteine machine; b is the quantized value of A.
FIG. 10 is a graph of sensitivity contrast of qPCR to verify the abundance of hmCLIP of the present invention versus prior art MICLIP pool.
FIG. 11 is a graph showing comparison of library effect of different amounts of RNA of hmCLIP; wherein A is the imaging result of the crosslinked ligation products with different RNA amounts on an adesaicing machine; b is the agarose gel running result of the PCR library with different RNA dosage.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
The process for preparing the thyroid cancer fine needle puncture paraffin specimen refers to [ Guo Yihe river, zhang Minfeng, meng Jiarong, yule ]. A method for preparing paraffin section by fine needle puncture suction cell mass [ J ]. J.Utility medicine, 2010,26 (21): 4034-4035 ].
Example 1
The application of hmCLIP in thyroid cancer fine needle puncture paraffin specimen sequentially comprises the following steps (the flow diagram is shown in fig. 1):
1. Obtaining Total RNA
Taking 3-5 paraffin specimens of thyroid cancer fine needle puncture, and extracting total RNA by QIAGEN RNeasy FFPE kit, wherein the specific method comprises the following steps:
1) Dewaxing paraffin sections: taking 3-5 paraffin slices, specifically according to the size of the slice tissue, placing the slices into a 1.5ml non-ribozyme EP tube, adding 1ml xylene, fully swirling for 10s, and centrifuging at the maximum rotation speed for 2min;
2) Removing the supernatant as much as possible, adding 1ml of absolute ethyl alcohol into the sediment, mixing uniformly by vortex, and centrifuging for 2min at the maximum rotation speed;
3) Removing the supernatant as much as possible, uncovering and standing at room temperature for 10min until the residual ethanol is volatilized;
4) 150ul PKD buffer (from QIAGEN RNeasy FFPE kit) was added and vortexed;
5) Adding 10ul proteinase K (from QIAGEN RNeasy FFPE kit), and mixing with gun head;
6) Incubating for 15min at 56 ℃ and incubating for 15min at 80 ℃;
7) Placing on ice for 3min and centrifuging for 15min with 20000 g;
8) Transferring the supernatant to a new tube, adding 16ul DNase Booster Buffer and 10ul DNase I (both from QIAGEN RNeasy FFPE kit), mixing upside down, and centrifuging briefly;
9) Incubating for 15min at room temperature;
10 320ul RBC buffer (from QIAGEN RNeasy FFPE kit) was added and thoroughly mixed;
11 720ul of 100% ethanol is added into the sample, and the mixture is mixed evenly by a gun;
12 700ul of the liquid was transferred to RNeasy MinElute spin column, >8000g centrifuged for 15s, and the paper adsorbed all of the liquid to the column;
13 500ul RPE buffer (from QIAGEN RNeasy FFPE kit) column wash, >8000g centrifuge 15s;
14 500ul RPE buffer (from QIAGEN RNeasy FFPE kit) column wash, >8000g centrifuge for 2min;
15 Opening the cover, and centrifuging at the maximum rotation speed for 5min to remove residual liquid as much as possible;
16 20ul nuclease-free water) was added, the mixture was centrifuged at maximum speed for 1min, and the product was collected as total RNA.
2. Acquisition of rRNA removal RNA
By Ribo-off TM rRNA depletion kit (Human/Mouse/Rat) (Northenzan) removes rRNA by:
1) 1ug total RNA was placed in a PCR tube, diluted to 11ul with nuclease-free water, and placed on ice for use.
2) The following reaction solutions were prepared in one PCR tube:
| rRNA Probe(H/M/R) | 1ul |
| Probe buffer | 3ul |
| Total RNA | 11ul |
| totals to | 15ul |
Lightly blowing and mixing by using a gun;
3) The samples were placed in a PCR instrument and operated as follows: 95 ℃ for 2min;95-22 ℃,0.1 ℃/sec;22 ℃ for 5min;
4) Adding 4ul RNase H buffer and 1ul RNase H into the reaction system, and incubating for 30min at 37 ℃;
5) 29ul DNase I buffer and 1ul DNase I are added into the reaction system, and the mixture is incubated for 30min at 37 ℃;
6) 110ul of Agencourt RNAClean XP Beads (Beckman) is added into the reaction solution, and the mixture is incubated on ice for 15min, so that the magnetic beads are fully combined with RNA;
7) Placing the sample on a magnetic rack, carefully removing the supernatant;
8) Rinsing twice with 200ul of 80% (v/v) ethanol;
9) Keeping the sample tracing in a magnetic frame, and uncovering the cover and drying the air for 5 to 10 minutes;
10 9ul of Nuclease-Free Water is added, the mixture is blown and evenly mixed, the mixture is kept stand at room temperature for 2min, the mixture is kept stand on a magnetic rack for 5min, and the supernatant is rRNA remote RNA.
3. RNA fragmentation:
RNA fragmentation system:
| component (A) | Volume (ul) |
| RNA(20ng/ul) | 9ul |
| Fragmentation reagent (10×) | 1ul |
| Total volume of | 10ul |
In the table, the fragmentation reagent (10×) was: 100ul 1M Tris-HCl (pH=7.4), 100ul 1M ZnCl 2 300ul Nuclease-free water (nucleic acid)-Free Water)。
The temperature and incubation time are as follows: 94 ℃ for 10s; the reaction was immediately stopped by the addition of EDTA (fragmented RNA was about 100nt long).
4. Co-immunoprecipitation and cross-linking
1) Reagent preparation
(1) Binding/low-salt buffer: comprises 50mM Tris-HCl (pH 7.4), 150mM NaCl and 0.5% (v/v) Igepal CA-630 (Sigma).
(2) High-Salt buffer: comprises 50mM Tris-HCl (pH 7.4), 1M NaCl, 1% (v/v) Igepal CA-630 and 0.1% (w/v) SDS (sodium dodecyl sulfate).
(3) 1x FastAP Buffer: comprises 10mM Tris-HCl (pH 7.5), 5mM MgCl 2 100mM KCl and 0.02% (v/v) Triton X-100.
(4) 5x PNK pH 6.5buffer: comprises 350mM Tris-HCl (pH 6.5) and 50mM MgCl 2 。
(5) 1x RNA Ligase buffer: comprises 50mM Tris-HCl (pH 7.5) and 10mM MgCl 2 。
(6) Wash buffer: comprises 20mM Tris-HCl (pH 7.4), 10mM MgCl 2 And 0.2% (v/v) Tween-20.
(7) High salt wash buffer: comprises 50mM Tris-HCl (pH 7.4), 1M NaCl, 1mM 0.5M EDTA (ethylenediamine tetraacetic acid), 1% (v/v) NP-40 (ethylphenyl polyethylene glycol), 0.1% SDS and 0.5% sodium deoxycholate (Sodium deoxycholate).
(8) Proteinase K reaction buffer: comprises 100mM Tris-HCl (pH 7.4), 50mM NaCl, 1mM EDTA (pH 8.0) and 0.2% (w/v) SDS.
Note that: the above concentrations are all the final system concentrations.
2) M6A immunoprecipitation of RNA
The following procedure was carried out using 200ng of RNA as the "IP (2) fraction" of the RNA fragment obtained in step 3.
IP sample operation:
(1) the RNA fragment was subjected to denaturation under the following conditions: placing the mixture on ice for 2-3 min at 75 ℃ for 5 min; incubating at 4 ℃ for 2 hours;
(2) precooling a 12-well plate on ice, transferring the reaction solution into the well plate, and using Spectroline UV 254nm 150mj/cm of instrument (model: XLE-1000 crosslinking instrument) 2 UV crosslinking at 254nm for 3 times, and shaking the pore plate at intervals for each time is sufficiently uniform;
(3) co-immunoprecipitation of beads with RNA-antibody complexes:
20ul protein A/G beads (Thermo, # 88802) were taken, washed twice with 500ul binding/low-salt buffer, resuspended with 100ul binding/low-salt buffer, and the resuspension was also added to the (2) cross-linked product and incubated for 2h at 4 ℃.
5. RNA linker ligation:
1) Washing the co-immunoprecipitated product obtained in the step 4 twice by using 900ul high-salt buffer; then washing twice with 900ul binding/low salt buffer; then washing twice with 500ul 1 XFastAP buffer;
2) Fastpap treatment:
| 10x FastAP Buffer(Thermo,EF0651) | 5ul |
| RNasin(Promega,N2111S) | 1ul |
| FastAP enzyme(Thermo,EF0651) | 4ul |
| Nuclease free water | 40ul |
| total volume of | 50ul |
Mixing, adding into sample, and homogenizing at 1200rpm and 37deg.C for 15min;
3) T4 PNK processing:
| 5x PNK pH 6.5Buffer | 30ul |
| RNasin | 2.5ul |
| T4 PNK(NEB,#M0210) | 3.5ul |
| 0.1M DTT (dithiothreitol) | 1.5ul |
| Nuclease free water | 112.5ul |
| Total | 150ul |
Mixing well, placing in Eppendorf Thermomixer: 37 ℃,15s 1400rpm,90s rest,20min; then standing on a magnetic rack for 1min, and removing the supernatant.
4) Cleaning magnetic beads:
(1) washing the product treated by the T4 PNK in the step 3) once by using 500ul Wash buffer (precooling at 4 ℃);
(2) adding 500ul Wash Buffer (pre-cooling at 4deg.C), placing on a magnetic rack, adding 500ul High salt Wash Buffer, and discarding supernatant;
(3) Adding 500ul cold High salt Wash buffer (pre-cooling at 4deg.C) suspended magnetic beads, placing on a magnetic rack, adding 500ul Wash buffer, and discarding supernatant;
(4) wash one pass with 500ul cold wash buffer;
(5) adding 500ul Wash Buffer suspended magnetic beads, placing on a magnetic frame, adding 300ul 1x RNA Ligase Buffer, and discarding supernatant;
(6) washing twice with 300ul 1x RNA Ligase Buffer;
5) The 3'ligation mater mix connection was configured on ice, the system was as follows:
gently mixing 3'ligation mater mix connection system with gun; it was then added to the sample after washing the beads in step 4) and placed in Eppendorf Thermomixer: 25℃and 15s 1200rpm,90s rest.
6) Washing off excess linker:
(1) placing the system connected in the step 5) on a magnetic rack to remove the supernatant;
(2) 500ul of pre-cooling (4 ℃) Wash Buffer is added, the magnetic beads are suspended and then placed on a magnetic frame, and the supernatant is discarded;
(3) adding 500ul of pre-cooled (4 ℃) Wash Buffer, placing on a magnetic rack, adding 500ul High salt Wash Buffer, and discarding the supernatant;
(4) washing with 500ul of pre-chilled (4 ℃) High Salt Wash Buffer;
(5) 500ul of pre-cooled (4 ℃) High Salt Wash Buffer suspended magnetic beads are placed on a magnetic rack, 500ul of Wash Buffer is added, and the supernatant is discarded;
(6) Wash Buffer was pre-cooled (4 ℃ C.) by 500ul twice.
7) Eluting the cross-linked RNA-antibody complex:
fresh configuration 1xLDS Sample Buffer:
| 4x Bolt TM LDS Sample Buffer(Thermo,#B0008) | 5ul |
| Nuclease free water | 13ul |
| 10x Reducing agent(Thermo,NP0004) | 2ul |
20ul of 1x LDS sample buffer was added to the beads washed free of excess linker in step 6) and heated at 70℃for 10min. The beads were separated, and the supernatant obtained was subjected to the next step.
8) Running glue and transferring:
the supernatant obtained in step 7) was loaded onto NuPAGE 4-12% gradient Bis-Tris Gels (thermo), buffer 1× MOPS SDS running buffer (thermo, cat: NP 0001), 200v,25min; after running the gel, the gel was transferred to a nitrocellulose membrane, and the buffer was transferred to a transfer buffer (thermo, cat# NP 00061) at 4℃for 400mA for 1 hour.
9) Isolating RNA in the crosslinked product:
placing the strip in the step 8) on an Odyssey bicolor infrared fluorescence imaging system for observation, imaging by means of an Odyssey infrared light emitting system, and observing the RNA joint connection condition of the crosslinked product by virtue of fluorescence signal intensity; the crosslinked product was cut off with a knife blade, the film was cut into strips 0.5-1 mm wide, and excess liquid was removed.
10 Proteinase K digestion):
the product isolated on the membrane of step 9) (i.e. antibody-RNA complex) was purified using protease K (Thermo Fisher Scientific;20 mg/mL) and incubated at 50 ℃ for 1h, the reaction system was formulated as follows:
| Proteinase K reaction Buffer | 200ul |
| Proteinase K | 10ul |
11 200ul of phenol chloroform isoamyl alcohol (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1) is added into the reaction liquid after the reaction in the step 10) for extraction, the temperature is 37 ℃, and the speed is 1400rpm for 10min.
12 After simple centrifugation of Heavy Phase Lock Gel tube (5 Prime, # 2302830), the reaction solutions of step 11) were all transferred to Heavy Phase Lock Gel tube, and the supernatant was collected by centrifugation (at room temperature, 13000rpm,2 min).
13 Dynabeads MyOne Silane (thermo, cat No.: 37002D) Recovery of RNA by magnetic beads:
(1) 10ul MyONE Silane beads (Thermo, # 37002D) was taken and the supernatant removed.
(2) The cells were washed once with 900ul of RLT buffer.
(3) Suspended with 600ul of RLT buffer, then about 200ul of the supernatant obtained in step 12) was added and vortexed.
(4) And (3) adding 20ul of 5M NaCl and 1230ul of 100% absolute ethyl alcohol (sigma) into the sample obtained in the step (3), mixing uniformly by vortex, and incubating for 15min at room temperature.
(5) Placing the sample obtained in the step (4) on a magnetic rack, removing the supernatant, adding 500ul of freshly prepared 80% (v/v) ethanol, suspending by using a gun, and transferring to a new tube.
(6) Separating magnetic beads from the sample obtained in the step (5) by using a magnetic frame, and removing the supernatant.
(7) Washing the sample obtained in the step (6) with 80% (v/v) ethanol twice for 30s each time, and removing the supernatant as much as possible.
(8) And (3) opening a tube cover of the sample obtained in the step (7), and drying at room temperature for 5min.
(9) The beads were suspended (RNA recovered) with 10ul Nuclease free water.
6. Input sample operation:
1) The RNA fragment obtained in the step 3 was prepared by taking 50ng of RNA as the "input (1) part", and the specific steps were as follows:
PNK treatment input RNA, shake incubation, conditions: 1200rpm,37C,20min; the system is as follows:
2) Dynabeads MyOne Silane (thermo, cat: 37002D) Recovery of RNA by magnetic beads:
(1) 10ul silane beads were removed from the supernatant.
(2) The cells were washed once with 900ul of RLT buffer.
(3) The beads were resuspended in 150ul RLT buffer to give a suspension.
(4) Adding the suspended magnetic beads obtained in the step (3) into the reaction system of the step (1), and vibrating and uniformly mixing;
(5) 5ul of 50% (w/v) PEG8000, 307.5ul of 100% ethanol are added into the step (4), mixed by shaking, and kept stand at room temperature for 15min.
(6) The supernatant was removed from the sample obtained in step (5), and the sample was washed twice with 80% (v/v) ethanol for 30 seconds each time to remove the supernatant.
(7) The sample obtained in step (6) was dried for 5min, added with 5ul Nuclease free water for solubilization, and the supernatant was transferred to a new tube.
3) The 3' end is connected with an RNA joint with fluorescent label:
the RNA recovered in step 2) was mixed with 1.5ul of 100% dmso, and 5nM of fluorescent-labeled RNA linker (sequence: 5 Phos/AUAUAGGNNNNNAGAUCGGAAGCGUCGUGUAG/3 AzidecN/, synthesized by IDT company), incubation at 65℃for 2min, ice for 1min, and addition of the following system, incubation at room temperature for 75min:
4) Dynabeads MyOne Silane recovery of adaptor-ligated RNA:
(1) 10ul silane beads were removed from the supernatant.
(2) The cells were washed once with 900ul of RLT buffer.
(3) The beads were resuspended in 63ul RLT buffer to give a suspension.
(4) Adding the suspended magnetic beads obtained in the step (3) into the reaction system of the step (3), and vibrating and uniformly mixing;
(5) adding 63ul of 100% ethanol into the step (4), blowing and mixing uniformly by using a gun, and standing for 15min at room temperature.
(6) The supernatant was removed from the sample obtained in step (5), and the sample was washed twice with 200ul of 80% (v/v) ethanol for 30 seconds each time to remove the supernatant.
(7) The sample obtained in step (6) was air-dried for 5min, 10ul Nuclease free water was added for solubilization, and the supernatant was transferred to a new tube (RNA after linker ligation was recovered).
7. Building a warehouse:
1) Reverse transcription reaction:
the RNA recovered in the above steps 5 and 6 was transferred to a PCR tube, and 0.5ul of reverse transcription primer AR17 (SEQ ID NO: ACACGACGCTCTTCCGA; IDT Co., ltd.) and 1ul of 10mM dNTP mix were added, respectively, and incubated at 65℃for 5 minutes, immediately transferred to ice for use, and the following system was added, and incubated at 55℃for 1 hour, and the reaction system was as follows:
2) ExoSAP-IT removes RNA:
(1) 3.5ul of ExoSAP-IT (Thermo, 78201.1. ML) was added to the reaction system of step 1), vortexed, centrifuged briefly, and incubated on a PCR instrument at 37℃for 15min.
(2) 1ul of 0.5M EDTA was added to the incubated sample in step (1), and the mixture was mixed with a gun.
(3) 3u 1M NaOH solution is added into the step (2), the mixture is mixed evenly by a gun, and the mixture is incubated for 12min at 70 ℃.
(4) To the sample after incubation in step (3) was added 3ul of 1M HCl solution for pH neutralization.
3) Dynabeads MyOne Silane (Thermo, cat: 37002D) Purifying and recovering cDNA:
(1) 10, ul MyONE Silane beads, the supernatant was removed.
(2) Washed once with 500ul 1x RLT buffer.
(3) Suspended with 93ul of RLT buffer and added to the reaction system obtained in step 2).
(4) 111.6 mu.l of 100% ethanol is added into the reaction system in the step (3), and the mixture is blown twice by a gun and incubated for 10min at 25 ℃.
(5) The incubated sample from step (4) was placed on a magnetic rack, the supernatant was removed, and washed twice with 300ul 80% (v/v) ethanol for 30s each time.
(6) And (3) simply centrifuging the sample subjected to alcohol washing in the step (5) by using a miniature centrifuge, separating magnetic beads by using a magnetic frame, and sucking residual ethanol as much as possible by using a gun.
(7) The tube cap was opened and dried at room temperature for 5min.
(8) 5ul Nuclease free water is added to the dried sample in the step (7) to suspend.
4) 3' linker ligation of cDNA:
the beads obtained in step 3) were added to 0.8 μl of rand3Tr345 (3' end cDNA linker, sequence: 5Phos/NNNNNNNNNNAGATCGGAAGAGCACACGTCTG/3 SpC/, synthesized by IDT company), 1 μl of 100% DMSO,1.5 μ l Nuclease free water, mixing with a gun blow, incubating at 75deg.C for 2min, ice for 2min, adding a ligase reaction system, shaking, and incubating at 25deg.C on a mixer for 15s 1400r.p.m,90s rest overnight. Wherein the ligase reaction system is as follows:
5) Dynabeads MyOne Silane purification recovery of cDNA ligation products:
(1) take 5ul MyONE Silane beads and remove the supernatant.
(2) The sample from which the supernatant was removed in step (1) was washed once with 500ul of RLT buffer.
(3) The sample obtained in step (2) was suspended with 60. Mu.l of RLT buffer and added to the reaction system after the enzyme ligation in step 4).
(4) 60 μl of 100% ethanol is added into the system of the step (3), and the mixture is stirred and mixed with a gun, and incubated for 5min at 25 ℃.
(5) The incubated sample from step (4) was placed on a magnetic rack, the supernatant was removed, and washed twice with 300ul 80% (v/v) ethanol for 30s each time.
(6) And (3) simply centrifuging the sample subjected to alcohol washing in the step (5) by using a miniature centrifuge, separating magnetic beads by using a magnetic frame, and sucking residual ethanol as much as possible by using a gun.
(7) The tube cap was opened and dried at room temperature for 5min.
(8) To the dried sample of step (7) was added 20 mu l Nuclease free water to suspend, and the supernatant was transferred to a new tube.
6) And (3) PCR amplification:
the reaction system is prepared as follows and then is put into a PCR instrument, and the temperature is 98 ℃ for 30s;98 ℃,15s,68 ℃,30s,72 ℃,40s, 16-20 cycles;72 ℃ for 5min; stored at 4℃and PCR was set.
| 2x Q5 PCR master mix | 25ul |
| PCR primer mix | 5ul |
| Purification of the recovered cDNA (i.e., step 5) ligation product | 20ul |
| Total | 50ul |
7) And (3) purifying a PCR product:
Adding 40ul Agencourt AMpure XP beads (Beckman, GN-A63880) to the PCR product (50 ul) obtained in step 6), incubating at room temperature for 5min, transferring to a magnetic rack, sucking the supernatant, then adding 10ul Agencourt AMpure XP beads, incubating at room temperature for 5min, transferring to the magnetic rack, and discarding the supernatant; washing the magnetic beads twice with 200ul of 80% (v/v) ethanol, and air-drying at room temperature for 3min; 35ul nuclease free water is added, the mixture is placed for 2min at room temperature and transferred to a magnetic rack, 30ul of supernatant is transferred to a new tube, the concentration is measured by using Qubit, and the measured concentration is 6-10 ng/ul. Respectively constructing an m6A-CLIP library (hmCLIP library) and an input library.
8. Sequencing on machine
1) Aligent Bioanalyzer 2100 quality control analysis.
2) High throughput sequencing was performed using the illumine corporation Hiseq X10 platform using PE150bp and index length 8bases (PE 150: pair end 150bp, representing a 150bp length of double ended sequencing; index length 8bases: adding an index with the length of 8bases on library in the library establishment process, and distinguishing different samples; wherein 8bases may be any of the following: the cgagtataat/tctccgga/aatgagcg/ggaatctc/ttctgaat/acgaattc/agcttcag/gcgctata) obtained about 30million reads per sample for subsequent data analysis, specific sequencing procedures and reagents were performed using illumine company standard procedures and kits, and sequencing results produced 100M reads, which were able to be aligned to more than 62% of human genome (Hg 19).
9. Analysis of results
(1) The detection and analysis result of the library obtained after the library is built by the invention through Agilent bioanalyzer 2100 is shown in figure 2.
(2) The bioinformatics analysis flow matched with hmCLIP analyzes the motif and distribution characteristics of m 6A:
(1) performing quality control on fastq files obtained by the illumine sequencing platform by using FASTQC;
(2) removing the 5 'linker sequence (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and the 3' linker sequence (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3') using a Cutadapt;
(3) PCR repeats were removed using FASTX; cutadapts remove random bases;
(4) aligning the processed sequence to the hg19 reference genome using Bowtie to obtain a SAM file;
(5) converting the SAM file into a BAM file using SAMTools;
(6) performing call peak on the BAM file by using exomepeak;
(7) performing bigwig file conversion on a BAM file by using Bam2bigwig, performing visual map viewing by using IGV, and FIG. 5 is an example of MYC genes in an hmCLIP library and a MICLIP library;
(8) the peak was subjected to a motif analysis using a Homer, see fig. 3A;
(9) the distribution profile was performed according to the peak position distribution on the transcript, see FIG. 3B;
to read with novolaign2b.pl and bedExt.pl ("novolaign2b.pl and bedExt.pl" represent published perl packages), respectively, see FIG. 4.
(3) An example of methylation of a single gene displayed by IGV is shown in FIG. 5 (MICLIP reference: linder B, grozhik AV, olaroerin-George AO, meydan C, mason CE, jaffrey SR. Single-nucleotide-resolution mapping of m A and m6Am throughout the transdome. Nature methods.2015;12 (8): 767-72.Doi:10.1038/nmeth.3453.PubMed PMID:26121403;PubMed Central PMCID:PMC4487409.).
(4) Sequencing was verified with QPCR for confidence:
(1) the sequencing result of step 9 (2) was verified (EEF 1A1 is a common control index for detecting m6A positive and negative, and peak was also identified in the results of step 9 (2) (6). Designing a pair of negative control primers (i.e., no specific peak of the targeting sequence occurs) and a pair of positive primers (i.e., specific peak of the targeting sequence exists) for the peak signal of the sequencing result of EEF1A 1;
negative control primer:
Forward：5’-GGATGGAAAGTCACCCGTAAG-3’；
Reverse：5’-TTGTCAGTTGGACGAGTTGG-3’；
positive control primers:
Forward：5’-CGGTCTCAGAACTGTTTGTTTC-3’；
Reverse：5’-AAACCAAAGTGGTCCACAAA-3’。
(2) the extracted RNA (see steps 1 and 2 of example 1) was fragmented in the same manner as step 3 of example 1, and divided into two parts, RNA fragment I and RNA fragment II.
(3) The RNA fragment I is denatured at 75 ℃ for 5min, placed on ice for 2-3 min, then incubated with m6A antibody (Abcam) at 4 ℃ for 2h, and incubated with IgG antibody as a control (CST, #7074P 2) and protein A/G beads (Thermo, # 88802) at 4 ℃ for 2h;
(4) Washing the magnetic bead antibody complex obtained in the step (3) with binding/low-salt buffer for 5 times, and removing the supernatant;
(5) digesting the magnetic bead antibody complex by using protease K (Thermo Fisher Scientific), and extracting RNA in the supernatant by using an ethanol precipitation method;
(6) and (3) performing conventional quantitative PCR on the two regions in EEF1A1 by using the RNA fragments obtained in the step (5) and the RNA fragment II, and normalizing by using the RNA fragment II, wherein the result is shown in FIG. 6.
Example 2
Reference to steps 1 to 5 in example 1 is different in that: the RNA and m6A antibodies in step 4 (co-immunoprecipitation and crosslinking) were different in crosslinking energy and time, and were divided into the following 6 groups of experiments:
(1) No UV crosslinking (named UV-);
(2) Using Spectroline UV 254nm The instrument (model: XLE-1000) is crosslinked with the following crosslinking energy: 150mj/cm 2 Number of crosslinks: 2 times (designated as 150 mj/cm) 2 *2)；
(3) Using Spectroline UV 254nm The instrument (model: XLE-1000) is crosslinked with the following crosslinking energy: 150mj/cm 2 Number of crosslinks: 4 times (designated as 150 mj/cm) 2 *4)；
(4) Using Spectroline UV 254nm The instrument (model: XLE-1000) is crosslinked with the following crosslinking energy: 300mj/cm 2 Number of crosslinks: 2 times (designated as 300 mj/cm) 2 *2)；
(5) Using Spectroline UV 254nm The instrument (model: XLE-1000) is crosslinked with the following crosslinking energy: 400mj/cm 2 Number of crosslinks: 2 times (named 400 mj/cm) 2 *2)；
(6) Using Spectroline UV 254nm The instrument (model: XLE-1000) is crosslinked with the following crosslinking energy: 1500mj/cm 2 Number of crosslinks: 2 times (named 1500 mj/cm) 2 *2)。
By enriching the cross-linked product with magnetic beads, connecting the cross-linked product with a joint with fluorescent marks after PNK treatment, washing off the redundant joint, separating by running 4% -12% NuPAGE gel, and collecting signals by an infrared instrument (Adesale), the result is shown in figure 7, and we find that: the crosslinking energy is 150mj/cm 2 When the number of crosslinking was 4, the signal intensity of the crosslinked product was the highest, indicating 150mj/cm 2 * The 4-condition crosslinking effect is the best.
Example 3
The process is identical to example 1 steps 1 to 5.9) with the difference that: in the case of RNA adaptor ligation in step 5, RNA adaptor ligation at 3 'end in step 6 and 3' adaptor ligation of cDNA in pool building in step 7, RNA Ligase High conc concentrations were high concentration Ligase (T4 RNA Ligase 1,High Concentration (NEB: M0437M)) and normal Ligase (T4 RNA Ligase, NEB: M0204S) (initial concentrations were 30,000U/ml and 10,000U/ml, respectively), the ligation efficiency and the reproducibility of hmCLIP were as shown in FIG. 8 (the reproducibility of hmCLIP was that of examples 1 to 5.9).
As can be seen from fig. 8: the ligation products obtained by ligating the fluorescently labeled adaptors using high concentrations of ligase are significantly higher than ligation product signals of conventional ligases and are also highly reproducible.
Example 4
The method is the same as in step 1 to step 5 of example 1, except that: recovery conditions after release of RNA from the membrane in step 5 (fluorescent-labeled RNA linker ligation) were varied and divided into the following 5 groups of experiments:
1. ethanol precipitation
Recovering RNA by ethanol precipitation, which comprises the following specific steps:
(1) 200ul of the proteinase K digested solution containing the target RNA obtained in the step 5 (method and step 10) is purified by phenol chloroform (method and step 11)), transferred to Heavy Phase Lock Gel tube, centrifuged, and the supernatant is taken (method and step 12)), and then 800ul of absolute ethanol and 20ul of 3M NaAC,1ul Glycogen (for nucleic acid precipitation) are added respectively, and the mixture is uniformly mixed and allowed to precipitate at-20 ℃ overnight (i.e., the operation of step 13) of example 1 is not performed);
(2) Centrifuging at maximum rotation speed (18,000 g) at 4deg.C for 20min, and discarding supernatant;
(3) Washing twice with 75% (v/v) ethanol, centrifuging at maximum rotation speed (18,000 g) at 4deg.C for 5min, and discarding supernatant;
(4) Dissolving back with 10ul DEPC water, shaking, mixing, taking 0.2ul of the solution, spotting on NC film, and observing on infrared instrument (Ordecel).
2. Zymo column (Zymo column cleanup; goods number: R1016)
RNA was recovered by the Zymo column method, and the method comprises the following steps:
(1) 400ul of RNA binding buffer (kit self-contained, zymo research) was taken;
(2) 600ul of absolute ethanol is added;
(3) Transferring the proteinase K lysate obtained in the step 5 (see step 10 in particular) into Zymo-Spin column, centrifuging for 30s, and discarding the waste liquid;
(4) 400ul RNA Prep Buffer (zymo research, cat# R1016) was added to the column, centrifuged for 30s, and the waste was discarded;
(5) 700ul RNA Wash Buffer (zymo research, cat# R1016) was added to the column, centrifuged for 30s, and the waste liquid was discarded;
(6) 400 and ul RNA Wash Buffer are added into the column, the column is centrifuged for 30s, and waste liquid is discarded;
(7) Thoroughly centrifuging for 2min;
(8) Transferring the column to a new tube, adding 10ul DEPC water for incubation for 1min, centrifuging for 30s, and collecting RNA solution;
(9) A solution of 0.2ul was spotted on the NC film and observed on an infrared ray apparatus (Aldeser).
3. silane magnetic beads
The recovered RNA was purified by Myone silane beads (Thermo, cat# 37002D) as follows:
(1) Taking 10ul MyOne silane beads, and removing the supernatant;
(2) Washing the beads with 900ul of 1×RLT buffer;
(3) Suspending the beads with 600ul of 1 XRLT buffer, then adding about 200ul of proteinase K lysate supernatant (obtained in step 12) to the beads, and vortexing;
(4) 1200ul of 100% absolute ethanol (sigma) was added;
(5) Vortex mixing, standing at room temperature for 15min;
(6) Placing the mixture on a magnetic rack, and removing a supernatant;
(7) Adding 500ul of 80% (v/v) ethanol for washing, removing the supernatant, and washing twice;
(8) Sucking the residual ethanol with a gun head;
(9) Opening a tube cover, and drying for 5min at room temperature;
(10) Adding 10ul DEPC water, suspending magnetic beads, and incubating for 1min;
(11) Separating magnetic beads by a magnetic frame, and transferring the supernatant to a new tube;
(12) A solution of 0.2ul was spotted on the NC film and observed on an infrared ray apparatus (Aldeser).
4、silane+NaCl
The method for RNA recovery by purification of Myone silane beads (+NaCl) was optimized and specifically comprises the following steps:
RNA was recovered by purification using Myone silane beads (Thermo, cat# 37002D), see above under group 3 experiments, with the difference: during incubation of the beads with proteinase K lysate, 20ul of 5M NaCl solution was added.
5、silane+PEG 8000
The method for purifying and recovering RNA by optimizing Myone silane magnetic beads (+PEG 8000) comprises the following specific steps:
recovered RNA was purified using Myone silane beads (Thermo Fisher Scientific) (Thermo, cat# 37002D), see above under group 3 experiments, with the difference: 20ul of 50% (w/v) PEG8000 was added during incubation of the beads with proteinase K lysate.
The results of the above 5 sets of experiments are shown in fig. 9: as can be seen from FIG. 9, the recovery of RNA was significantly higher when NaCl or PEG8000 was added to the silane bead purification system than in the other groups.
Example 5
1. Sensitivity comparison experiment of hmCLIP and MICLIP library enrichment
The cDNA ligation product obtained by purification and recovery in example 1, step 7.5) was purified and purified using the methods previously reported (reference: linder B, grozhik AV, olarolin-George AO, meydan C, mason CE, jaffrey SR. Single-nucleotide-resolution mapping of m A and m6Am throughout the transcriptame. Nature methods.2015;12 (8) 767-72.Doi:10.1038/nmeth.3453.PubMed PMID 26121403; pubMed Central PMCID PMC 4487409.) cDNA from MICLIP was compared for library abundance by quantitative PCR. The results are shown in FIG. 10:
2. library effect comparison experiment of different RNA amounts of hmCLIP
(1) The method is the same as in step 1 to step 5.9 of example 1, except that: the RNA amounts in step 4.2) were different, respectively 0.1ug, 1ug, 10ug, proceeding to step 5.9), and the imaging results of the cross-linked ligation products of different RNA amounts on the adesaicing machine are shown in FIG. 11A; as can be seen from the figure, the signal at 0.1ug of RNA can still be captured, although the signal of the resulting cross-linked product-RNA adaptor ligation product will decrease with decreasing RNA usage.
(2) The samples (RNA amounts different) from step (1) were each continued to step 7.6 according to the method of example 1, while a blank control containing no template was set, and the PCR amplification product was used for agarose gel running, and the results are shown in FIG. 11B. From the figure, it can be seen that a clear library can still be obtained when the RNA amount is 0.1ug, so that hmCLIP library construction can reduce the RNA amount to 0.1ug.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
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Claims (4)
1. A method for detecting RNA m6A with high throughput and high sensitivity single base resolution for non-disease diagnosis purposes, comprising the steps of:
(1) Preparation of RNA samples for DerRNA
Extracting total RNA in a sample, and then removing rRNA in the total RNA to obtain an RNA sample;
(2) RNA fragmentation
Fragmenting the RNA sample obtained in the step (1) to obtain RNA fragments, dividing the RNA fragments into 2 parts, and naming the RNA fragments as RNA fragments I and II;
(3) Co-immunoprecipitation and cross-linking
Performing heat denaturation treatment on the RNA fragment I obtained in the step (2), and then reacting with an antibody specifically recognizing m6A locus to obtain a reaction solution; then crosslinking the reaction solution under the ultraviolet condition to obtain a crosslinked complex; adding protein A/G beads into the cross-linked complex for co-immunoprecipitation to obtain an co-immunoprecipitation product; wherein, the conditions of crosslinking are: 150mj/cm 2 UV crosslinking of 254 nm 4 times, or 300mj/cm 2 UV crosslinking of 254 nm 2 times, or 400mj/cm 2 UV crosslinking of 254, nm 2 times;
(4) RNA adaptor ligation
(1) Connecting the co-immunoprecipitation product obtained in the step (3) with a fluorescent labeling joint at the 3' -end of RNA, washing off redundant joints after connection, carrying out denaturation elution, and carrying out electrophoresis separation to obtain a crosslinked connection product; then adding proteinase K into the cross-linked connection product for digestion, and recovering RNA by using silane magnetic beads and NaCl or silane magnetic beads and PEG 8000 after digestion to obtain purified RNA;
(2) Connecting the RNA 3' terminal joint of the RNA fragment II obtained in the step (2) and recovering to obtain RNA connected by the joint;
(5) Building a warehouse
Reverse transcription of the purified RNA obtained in step (4) (1) into cDNA, and then 3' -terminal adaptor ligation of the cDNA to obtain adaptor-ligated cDNA; carrying out PCR amplification and purification on the cDNA connected with the connector to obtain a PCR product, namely constructing an hmCLIP library; replacing the RNA after the linker connection obtained in the step (4) (2) with the purified RNA obtained in the step (4) (1), and constructing an input library according to the same operation steps;
(6) Sequencing
Sequencing the hmCLIP library and the input library constructed in the step (5) by using a high-throughput sequencing platform;
the mass of the RNA fragment I in the step (2) is more than or equal to 100 ng;
the mass of the RNA fragment II in the step (2) is more than or equal to 10ng;
the antibody specifically recognizing the m6A site described in step (3) is an m6A antibody;
the T4 RNA Ligase used for the adaptor ligation in step (4) is T4 RNA Ligase 1, high Concentration;
the dosage of the T4 RNA ligase is calculated according to the addition of 2000U/ml of the final concentration of the T4 RNA ligase in a reaction system;
The 3' -end fluorescence labeling linker sequence of the RNA in the step (4) is as follows:
5 Phos/AUAUAGGNNNNNAGAUCGGAGAGGUGUGUAG/3 AzidecN/; wherein, N: a, c, g, or u; phos: modification of phosphate groups; azide: azide modification;
the dosage of NaCl in the step (4) (1) is calculated according to the addition of 50nmol/L of the final concentration of the NaCl in the reaction system;
the dosage of the PEG 8000 in the step (4) (1) is calculated by adding the PEG 8000 according to the final concentration of the PEG 8000 in a reaction system of 0.05 g/ml;
the 3' -terminal linker sequence described in step (5) is as follows:
5Phos/NNNNNNNNNNAGATCGGAAGAGCACACGTCTG/3SpC3/; wherein, N: a, c, g, or u; phos: modification of phosphate groups; spC3: spacers modification C3, preventing the joint from self-connecting;
the primer sequences required for reverse transcription described in step (5) are as follows: 5'-ACACGACGCTCTTCCGA-3'.
2. The method for high-throughput high-sensitivity single base resolution detection of RNA m6A of claim 1, wherein:
the mass of the RNA fragment I in the step (2) is more than or equal to 200 and ng;
the mass of the RNA fragment II in the step (2) is more than or equal to 50ng.
3. The method for high-throughput high-sensitivity single base resolution detection of RNA m6A of claim 1, wherein:
The thermal denaturation treatment conditions described in the step (3) are: treating at 75 deg.c for 5min, and then setting on ice for 2-3 min;
the conditions for co-immunoprecipitation described in step (3) are: incubating at 4 ℃ for 2 h;
the electrophoresis separation in the step (4) (1) is carried out by adopting 4% -12% NuPAGE;
the silane magnetic beads in the step (4) (1) are Dynabeads MyOne Silane magnetic beads;
the purification in the step (5) is performed by adopting Agencourt AMpure XP beads;
the high throughput sequencing platform described in step (6) is a Hiseq X10 platform from illuminea.
4. Use of the method for detecting RNA m6A with high throughput and high sensitivity single base resolution for non-disease diagnosis purposes according to any one of claims 1 to 3 for detecting RNA m6A, characterized in that: the application field is the non-disease diagnosis and treatment field.
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