MeRIP-seq (Methyled RNA Immunoprecipitation Sequencing), a high-throughput sequencing technique that combines ChIP-Seq and RNA-Seq methodologies, is utilized for the thorough analysis of RNA methylation modifications. In this article, we delve into the intricate operational procedures of MeRIP-seq.
This technique harnesses the power of immunoprecipitation to enrich for methylated RNA fragments, subsequently subjecting them to next-generation sequencing for comprehensive profiling. For a more in-depth understanding of the technical principles underlying MeRIP-seq, refer to"Principle and Applications of MeRIP-seq."
Illustration of MeRIP-Seq Technique(Meng J et al., 2014)
The following are the detailed steps of the protocol：
1. Material preparation
- Cell or tissue samples: for example, neuronal cells extracted from mouse brains, processed Arabidopsis leaves, rapidly frozen to maintain RNA integrity.
- Antibody: Select antibodies that target specific methylation markers, such as m6A antibodies.
- Lysis buffer: Lysis buffer containing RNase inhibitors, such as TRIzol or RIPA buffer.
2. RNA extraction
- RNA extraction: Total RNA is extracted from cell or tissue samples, typically using TRIzol or similar assay kits to ensure RNA quality and integrity.
3. RNA Fragmentation
- Select RNA samples: Use total RNA samples after extraction to ensure that their quality and concentration meet the experimental requirements.
- Enzymatic cleavage method: Use a specific RNase (e.g. RNase I or other enzymes) to enzymatically cleave the RNA under appropriate conditions. The concentration of the enzyme and the reaction time need to be optimised to obtain RNA fragments of the desired length (typically 100-300 nt).
- Thermal shearing (optional): RNA fragmentation can sometimes be achieved by high-temperature treatment (e.g., heating to 70-80°C for a few minutes). This method is usually not precise enough, but can be a quick option in some cases.
- Purification after fragmentation: After fragmentation, uncut long-stranded RNA and enzymes need to be removed by column purification or agarose gel electrophoresis to obtain pure RNA fragments.
- Concentration and quantification: Concentrate the RNA fragments using ethanol precipitation or other methods, and quantify using a spectrophotometer or fluorescence to ensure that the concentration of RNA is suitable in subsequent steps.
- Precautions: RNA degradation should be avoided when performing RNA fragmentation, so it is necessary to keep the samples at low temperatures and use RNase inhibitors throughout.The length of the fragments will affect the results of subsequent library construction and sequencing, and therefore needs to be optimised for the specific research purpose.RNA fragmentation can ensure the best results in subsequent m6A enrichment and sequencing.
4. RNA immunoprecipitation
- Antibody Incubation: Mix the fragmented RNA with an antibody specific for the m6A modification and incubate at 4°C for a period of time (usually 1-2 hours) to ensure that the antibody binds sufficiently to the target RNA.
- Add protein A/G agarose beads: Pre-equilibrated Protein A or G agarose beads are added to capture the antibody-RNA complexes, which are typically incubated for a further 30 minutes to 1 hour at low temperature.
5. Washing and dissociation
Wash step:
- After completing the antibody incubation, centrifuge the agarose beads containing the antibody-RNA complexes using a centrifuge, usually set at a lower speed (e.g., 2000-3000 rpm) for 5-10 minutes to allow the beads to settle to the bottom.
- Carefully remove the supernatant and avoid disturbing the beads to maintain the integrity of the antibody-RNA complex.
- Add an appropriate amount of wash buffer (usually a salt-containing buffer such as PBS or salt-containing Tris buffer) to the beads to help remove non-specifically bound RNA and protein.
- Mix gently to ensure that the beads are well-covered by the wash buffer before centrifuging to re-sediment the beads.
- Repeat the above washing process several times (3-5 times is generally recommended) to increase the thoroughness of the wash and to ensure removal of all non-specifically bound components.
Dissociate the RNA:
- Prepare an appropriate elution buffer, which can usually be a buffer containing a high salt concentration or denaturing conditions (e.g., a buffer heated to 65°C).
- Add the elution buffer to the washed beads, mix gently to ensure that the buffer completely covers the beads, and then incubate for a period of time (e.g., 5-10 minutes) at a suitable temperature to promote RNA dissociation.
- Perform centrifugation and transfer the eluted supernatant into a new tube, which here contains the enriched m6A modified RNA.
6. RNA purification
- The eluted RNA is subjected to further purification steps, such as column purification or phenol-chloroform extraction, to remove residual proteins and impurities and to obtain purified methylated RNA, which is ready for subsequent sequencing or analysis.
7. Library construction
- Junction addition at the 5' end: Addition of a specific junction sequence to the 5' end of a purified RNA fragment, usually using RNA junction ligase. This step usually requires the addition of a 'cap' structure to facilitate subsequent reverse transcription and sequencing.
- Addition of a splice at the 3' end: For the 3' end, the corresponding junction sequence is also added. This step usually involves polyadenylation to ensure that the junction can bind efficiently.
- Reverse transcription synthesis of cDNA: The RNA fragment with the junction attached is transcribed into complementary DNA (cDNA) using reverse transcriptase. This process usually requires primers, such as random primers or oligo dT primers, to ensure specificity and efficiency of reverse transcription.
- PCR amplification:PCR amplification of the synthesised cDNA is performed using appropriate primers to increase the concentration and specificity of the library. In this process, amplification can be optimised using specific primers containing sequencing primers.
- Library purification:Residual primers, dNTPs and other impurities in the PCR amplification reaction are removed by column purification or magnetic bead purification to ensure the purity of the final library.
8. Sequencing
- Library preparation: Ensure that the constructed library is purified and quantified to meet the requirements of the sequencing platform. Typically, the concentration of the library needs to be within a specific range (e.g., 10 nM to 20 nM).
- Library spiking: According to the requirements of the sequencing platform, the library samples are diluted according to the recommended ratio and added to the sequencing chip or flow cell. Different platforms may have different spiking methods.
- Sequencing primer addition: Depending on the sequencing technology used (e.g. Illumina, Ion Torrent, etc.), specific sequencing primers are added. These primers will bind to the library junction to initiate the sequencing reaction.
- Select the type of sequencing: Depending on the experimental design, choose the appropriate sequencing mode, such as Single-end sequencing (Single-end sequencing) or Paired-end sequencing (Paired-end sequencing). Paired-end sequencing can obtain richer information and is usually used for the analysis of complex samples.
- Perform a sequencing reaction: Starting the sequencer for sequencing. This process typically varies depending on the sequencing platform selected and may include cyclic synthetic sequencing or fluorescently labelled sequencing methods.
- Data Collection: During the sequencing process, the raw data generated is collected in real time, including sequence information for each base and its mass fraction. The sequencer automatically records this information for subsequent analysis.
- Data processing: Upon completion of sequencing, special software is used for preliminary data processing, including removal of low-quality sequences, clipping of splice sequences, and filtering of sequences.
- Storage and Analysis: Processed data are stored in a secure database for subsequent bioinformatics analysis. Multiple tools can be used for data comparison, identification of m6A modification sites and functional analysis.
Data analysis
Learn more: MeRIP-seq Analysis
- Data preprocessing: Quality control: use tools (e.g. FastQC) to check the quality of raw sequencing data, including sequencing quality score, GC content, and sequence length distribution.
- Removal of low-quality sequences: Remove low-quality reads according to a set threshold (e.g. Q-score) to improve the accuracy of subsequent analyses.Use software (e.g. Cutadapt or Trimmomatic) to remove splice sequences that may remain during sequencing to ensure that only pure RNA sequences are used in the analysis.
- Comparison: The cleaned sequences are compared to a reference genome (e.g., human genome GRCh38). Commonly used alignment tools include HISAT2, STAR or Bowtie2.Optionally, a multiple alignment strategy can be used during the alignment process to improve the identification of m6A modification sites.
- Peak calling: Use specific peak calling tools (e.g. MACS2) to identify regions enriched for m6A modifications. These tools can perform statistical analyses to identify significant peaks of enrichment based on the compared reads.
- Functional annotation: The identified m6A-enriched regions are cross-analysed with known gene information and functionally annotated using annotation databases (e.g. GENCODE or Ensembl) to understand their potential role in gene regulation.
- Differential analysis: Samples from different conditions are compared and statistical methods (e.g. DESeq2 or edgeR) are used to identify m6A modification sites that are significantly different between treatment groups.
- Visualisation: Graphical presentation of the analysis results using visualisation tools (e.g. IGV, ggplot2 or pheatmap), including distribution maps of m6A modifications, heatmaps and genome browser views.
- Biological interpretation: Based on the analysis results and in conjunction with existing literature, explore the effects of m6A modifications on specific gene expression, cellular functions and biological processes, propose relevant hypotheses and design follow-up experiments to verify them.
- Data archiving and sharing: The processed data and analysis results should be reasonably archived and considered for sharing in public databases (e.g., GEO or ArrayExpress) to facilitate scientific communication and follow-up research.
References:
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Zeng Y, Wang S, Gao S, Soares F, Ahmed M, Guo H, Wang M, Hua JT, Guan J, Moran MF, Tsao MS, He HH."Refined RIP-seq protocol for epitranscriptome analysis with low input materials."PLoS Biol. 2018;16(9):e2006092.
- Govindan G, Sunkar R."MeRIP-Seq for Identifying Stress-Responsive Transcriptome-Wide m6A Profiles in Plants."Methods Mol Biol. 2024;2832:47-55.
