Dec 25, 2025
m6A RNA immunoprecipitation (MeRIP)
- Xin Xu1,
- Yujuan Wang1
- 1Princess Margaret Cancer Center
Protocol Citation: Xin Xu, Yujuan Wang 2025. m6A RNA immunoprecipitation (MeRIP). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzenmrvx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 21, 2025
Last Modified: December 25, 2025
Protocol Integer ID: 235602
Keywords: m6A, RNA methylation, MeRIP, MeRIP-seq, epitranscriptomics, RNA modification, RNA immunoprecipitation, cancer, transcriptome profiling, rna immunoprecipitation, enrichment of m6a, m6a, using fragmented total rna, total rna, fragmented total rna from cell line, modified rna, merip, antibody, purification for downstream library preparation, purification
Disclaimer
This protocol is provided for research use only and may require optimization depending on experimental conditions.
Abstract
This protocol describes m6A RNA immunoprecipitation (MeRIP) using fragmented total RNA from cell lines, followed by antibody-based enrichment of m6A-modified RNA and purification for downstream library preparation.
Guidelines
- Keep IGEPAL concentration consistent across buffers (0.1%) to minimize nonspecific binding while maintaining bead handling.
- During column cleanup, residual ethanol is a frequent cause of poor downstream enzymatic reactions; do the long dry spin.
Materials
**Reagents and materials**
**Core reagents**
- Total RNA (cell line), 5 µg
- Protein A magnetic beads (Thermo Fisher, 10002D**), 30 µL per IP
- Protein G magnetic beads (Thermo Fisher, 10004D**), 30 µL per IP
- Anti-m6A antibody (NEB, E1610S), 1 µL per IP (per your manuscript)
- RNasin Plus RNase Inhibitor (Promega, N2611)
- RNA Fragmentation Buffer (see recipe; your manuscript: 100 mM Tris-HCl pH 7.4; 100 mM ZnCl2)
- 0.5 M EDTA
- Ethanol (100% and 70%-75%)
- RNeasy Mini Kit (QIAGEN, 74106**) - for RLT and RPE**
- RNeasy MinElute spin columns (as used in your lab protocol for final cleanup)
**meRIP-Seq Protocol**
- Magnetic rack for 1.5 mL tubes
- End-over-end rotator (4°C)
- Thermomixer or rotator for washes (4°C)
- Optional QC: TapeStation High Sensitivity RNA ScreenTape
**Buffers (prepare RNase-free)**
**1x IP buffer (used for bead wash and IP)
- 10 mM Tris-HCl, pH 7.5
- 150 mM NaCl
- 0.1% IGEPAL CA-630
**5x IP buffer stock (optional but convenient for making the IP mixture)
Recipe example (30 mL): 4.5 mL 5 M NaCl + 1.5 mL 1 M Tris-HCl pH 7.5 + 0.15 mL IGEPAL + water to 30 mL.
**1x low-salt wash buffer**
- 10 mM Tris-HCl, pH 7.5
- 50 mM NaCl
- 0.1% IGEPAL CA-630
**1x high-salt wash buffer**
- 10 mM Tris-HCl, pH 7.5
- 500 mM NaCl
- 0.1% IGEPAL CA-630
Troubleshooting
Before start
- Use RNase-free tubes, tips, and reagents throughout.
- Perform all steps on ice or at 4°C unless otherwise indicated.
- Pre-chill rotators and magnetic racks.
- Prepare all buffers with nuclease-free water.
- Add RNase inhibitor immediately before use.
Procedure
Aliquot 5 µg total RNA into an RNase-free tube.
Adjust volume with nuclease-free water to 18 µL.
Add 2 µL RNA Fragmentation Buffer (final composition consistent with your manuscript: 100 mM Tris-HCl pH 7.4; 100 mM ZnCl2).
Incubate at 70°C until theaverage size is ~180 nt (optimize time empirically for your setup).
Immediately place on ice. Add EDTA to chelate Zn2+ and stop fragmentation.
Ethanol precipitate the fragmented RNA, and resuspend fragmented RNA in RNase-free water.
Save 10% of fragmented RNA as input (store at -80°C).
Optional: assess fragmentation size distribution by TapeStation.
Vortex beads to resuspend.
Add 30 µL Protein A beads + 30 µL Protein G beads to a tube.
Add 1 mL cold 1x IP buffer, invert to mix, place on magnet, remove supernatant.
Repeat once (total 2 washes).
Resuspend beads in 500 µL 1x IP buffer.
Add 1 µL anti-m6A antibody (NEB E1610S).
Rotate at 4°C for at least 6 h.
Wash the beads twice with 1 mL cold 1x IP buffer (magnet separate each time).
After the second wash, resuspend beads in 500 µL of IP reaction mixture (next section).
Immunoprecipitation (2 h at 4°C)
2h
In a final volume of 500 µL, combine:
Fragmented RNA (from Section A; excluding the 10% input)
100 µL 5x IP buffer (to achieve 1x in reaction)
5 µL RNasin Plus (Promega N2611)
Add nuclease-free water to 500 µL total
Add this mixture to the antibody-coupled beads.
Rotate 2 h at 4°C.
2h
Wash beads (stringent low/high-salt scheme)
1h 25m
Wash 1x IP buffer (2x)
Add 1 mL cold 1x IP buffer, rotate 10 min at 4°C, magnet separate, discard supernatant.
Repeat once (total 2 washes).
25m
Wash low-salt buffer (2x)
Add 1 mL cold low-salt buffer, rotate 10 min at 4°C, magnet separate, discard supernatant.
Repeat once.
25m
Wash high-salt buffer (2x)
Add 1 mL cold high-salt buffer, rotate 10 min at 4°C, magnet separate, discard supernatant.
Repeat once.
25m
Final quick spin (optional)
Briefly spin tube (5-10 s) to collect residual liquid.
Place on magnet and remove remaining wash buffer carefully.
10m
Elution and RNA purification (RNeasy MiniElute)
22m
Elute from beads with RLT
Add 200 µL RLT buffer directly to beads.
Mix and incubate 2 min at room temperature.
Place on magnet and transfer supernatant to a new tube.
5m
Bind to column
Add 400 µL 100% ethanol to the eluate, mix well.
Load onto an RNeasy MiniElute spin column; centrifuge at 12,000 rpm, 1 min, 4°C.
If needed, load in multiple passes until the full volume is processed.
5m
Wash column
Wash with 500 µL RPE once.
Wash with 500 µL 80% ethanol once.
Spin full speed 5 min at 4°C to remove residual ethanol.
10m
Elute IP RNA
Elute with 14 µL ultrapure H2O.
Store at -80°C.
2m
Optional checkpoints (recommended)
4h
Fragmentation QC: TapeStation HS RNA to confirm ~180-200 nt distribution.
IP efficiency QC: RT-qPCR for known methylated vs negative regions.
4h
Notes and common pitfalls
Fragmentation time is the main driver of final fragment size. Once you have a time that yields ~180 nt for 5 µg in your thermocycler, keep it constant across samples.
Keep IGEPAL concentration consistent across buffers (0.1%) to minimize nonspecific binding while maintaining bead handling.
During column cleanup, residual ethanol is a frequent cause of poor downstream enzymatic reactions; do the long dry spin.