Target-Specific Profiling of RNA m5C Methylation Level Using Amplicon Sequencing

Tennille Sibbritt; Ulrike Schumann; Andrew Shafik; Marco Guarnacci; Susan J Clark; Thomas Preiss

doi:10.1007/978-1-0716-1851-6_21

Target-Specific Profiling of RNA m⁵C Methylation Level Using Amplicon Sequencing

Methods Mol Biol. 2022:2404:375-392. doi: 10.1007/978-1-0716-1851-6_21.

Authors

Tennille Sibbritt^#¹, Ulrike Schumann^#¹, Andrew Shafik¹, Marco Guarnacci¹, Susan J Clark^{2

3}, Thomas Preiss^{4

5}

Affiliations

¹ Department of Genome Sciences, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
² Epigenetics Research Laboratory, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia.
³ St. Vincent Clinical School, University of New South Wales, Sydney, NSW, Australia.
⁴ Department of Genome Sciences, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. thomas.preiss@anu.edu.au.
⁵ Victor Chang Cardiac Research Institute, Sydney, NSW, Australia. thomas.preiss@anu.edu.au.

^# Contributed equally.

PMID: 34694621
DOI: 10.1007/978-1-0716-1851-6_21

Abstract

Mapping the position and quantifying the level of 5-methylcytosine (m⁵C) as a modification in different types of cellular RNA is an important objective in the field of epitranscriptomics. Bisulfite conversion has long been the gold standard for the detection of m⁵C in DNA, but it can also be applied to RNA. Here, we detail methods for bisulfite treatment of RNA, locus-specific PCR amplification, and detection of candidate sites by sequencing on the Illumina MiSeq platform.

Keywords: 5-methylcytosine; Bisulfite conversion; Epitranscriptomics; MiSeq; Next-generation sequencing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

5-Methylcytosine
High-Throughput Nucleotide Sequencing
Methylation
RNA / genetics
Sequence Analysis, DNA*

Substances

RNA
5-Methylcytosine