High-Resolution Mapping of N6-Methyladenosine Using m6A Crosslinking Immunoprecipitation Sequencing (m6A-CLIP-Seq)
N6-Methyladenosine, an abundant chemical modification in mRNA, plays crucial roles in regulating gene expression and biological processes. Research on m6A and its functions has progressed rapidly in the past few years, aided substantially by advances in high-throughput sequencing-based methods to profile m6A along the transcriptome. We present here a protocol for m6A crosslinking immunoprecipitation sequencing (m6A-CLIP-seq), which profiles m6A on mRNA at high resolution from as little as 1 μg of poly(A)-selected mRNA.
Key wordsN6-Methyladenosine Transcriptome Methylome Affinity purification Sequencing
We thank Caroline Vissers, Boxuan Zhao, and Allen Zhu for helpful comments. P.J.H. is supported by the NIH Medical Scientist National Research Service Award T32 GM007281. C.H. is supported by National Institutes of Health grants GM071440 and HG008935. C.H. is an investigator of the Howard Hughes Medical Institute.
- 2.Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vagbo CB, Shi Y, Wang WL, Song SH, Lu Z, Bosmans RP, Dai Q, Hao YJ, Yang X, Zhao WM, Tong WM, Wang XJ, Bogdan F, Furu K, Fu Y, Jia G, Zhao X, Liu J, Krokan HE, Klungland A, Yang YG, He C (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell 49(1):18–29. https://doi.org/10.1016/j.molcel.2012.10.015 CrossRefPubMedGoogle Scholar
- 6.Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485(7397):201–206. https://doi.org/10.1038/nature11112 CrossRefGoogle Scholar
- 9.Chen K, Lu Z, Wang X, Fu Y, Luo GZ, Liu N, Han D, Dominissini D, Dai Q, Pan T, He C (2015) High-resolution N(6)-methyladenosine (m(6)A) map using photo-crosslinking-assisted m(6) A sequencing. Angew Chem Int Ed Engl 54(5):1587–1590. https://doi.org/10.1002/anie.201410647 CrossRefPubMedGoogle Scholar