Identifying the m6A Methylome by Affinity Purification and Sequencing

Phillip J Hsu; Chuan He

doi:10.1007/978-1-4939-7213-5_3

Identifying the m⁶A Methylome by Affinity Purification and Sequencing

Methods Mol Biol. 2018:1649:49-57. doi: 10.1007/978-1-4939-7213-5_3.

Authors

Phillip J Hsu^{1

2

3}, Chuan He^{4

5

6}

Affiliations

¹ Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 E. 57th Street, Chicago, IL, USA.
² Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA.
³ Committee on Immunology, The University of Chicago, Chicago, IL, USA.
⁴ Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 E. 57th Street, Chicago, IL, USA. chuanhe@uchicago.edu.
⁵ Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA. chuanhe@uchicago.edu.
⁶ Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA. chuanhe@uchicago.edu.

PMID: 29130189
DOI: 10.1007/978-1-4939-7213-5_3

Abstract

N ⁶-methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic mRNA, and is newly emerging as a key posttranscriptional mRNA regulator. Recent research has uncovered insight into the location and function of m⁶A sites on a large scale, in part due to the transcriptome-wide identification of m⁶A sites by high-throughput sequencing (m⁶A-seq). Here, we present a protocol for m⁶A-seq, which maps the m⁶A methylome by affinity purification and sequencing.

Keywords: Affinity purification; Methylome; N 6-methyladenosine; Sequencing; Transcriptome.

MeSH terms

Adenosine / analogs & derivatives*
Adenosine / metabolism
Antibodies / metabolism
Chromatography, Affinity / methods*
Gene Library
High-Throughput Nucleotide Sequencing / methods*
Metabolome*
Methylation
Quality Control
RNA / isolation & purification
RNA / metabolism

Substances

Antibodies
RNA
N-methyladenosine
Adenosine