m^6 A-seq consists of affinity purification of m^6 A-marked RNA
fragments using an m^6 A-specific antibody, followed by high-
throughput sequencing and identification of m^6 A-enriched sites.
The affinity purified fragments are around 100–150 nucleotides
long, and may contain the m^6 A at any location. Analysis of the
overlap between the fragments and the genome, as well as a com-
parison of fragments from affinity purified (“IP”) and unpurified
(“input”) samples, allows organization of the fragments into
“peaks.” Identification of the midpoint of the peak helps to localize
the m6A. Because m^6 A is typically present on a consensus motif of
DRACH (D¼A, G, U; R¼A, G; H¼A, C, U), identification of
this motif may help further identify the exact location of the m^6 A
[5, 7].
m^6 A-seq is nonetheless limited in its ability to pinpoint m^6 Aat
single-nucleotide resolution. Most DRACH sites across the
genome do not contain m^6 A, and peaks may not have a clear
midpoint due to uneven fragmentation or clusters of m^6 A residues.
Recent updates to m^6 A-seq have improved on this limitation. A
photocrosslinking-assisted m^6 A-sequencing strategy, PA-m^6 A-seq,
provides a higher resolution transcriptome-wide map of m^6 A, and
miCLIP, a method based on UV-induced antibody-RNA crosslink-
ing, provides single-nucleotide resolution localization of m^6 A
across the transcriptome [8, 9]. Furthermore, m^6 A-seq does not
provide a stoichiometric analysis of the fraction of modified tran-
scripts. A recent improvement, m^6 A-LAIC-seq, quantitatively iden-
tifies the proportion of a m^6 A-containing transcripts of a given gene
(9, reviewed in10). Although these limitations of m^6 A-seq have
been met, a remaining challenge and area of active research is the
development of a method to sequence m6A in low-abundance
samples or in single cells.
Here we present our protocol for m^6 A-seq. Similar protocols
have been published elsewhere [6, 12–14]. A detailed bioinformat-
ics analysis pipeline has been published by the author of the original
protocol [12].2 Materials
Prepare all solutions using DEPC-treated nuclease-free water and
molecular grade reagents. Be sure to prepare all reagents supple-
mented with SUPERase inhibitor or BSA freshly, as integrity may
be compromised if left at 4CorRT.2.1 Reagents 1. Cultured cells or tissues as a source of RNA. Any cell line or
tissue is suitable for this procedure. As a reference, one conflu-
ent 10 cm plate of HeLa cells provides ~100μg of total RNA,
2–5% of which is mRNA. At least 1μg of polyA-selected mRNA
is required.
50 Phillip J. Hsu and Chuan He