Transcriptome-Wide Detection of 5-Methylcytosine by Bisulfite Sequencing

Methods Mol Biol. 2017:1562:123-142. doi: 10.1007/978-1-4939-6807-7_9.

Abstract

While low-throughput RNA bisulfite sequencing is the method of choice to assess the methylation status of specific cytosines in candidate RNAs, the combination of bisulfite treatment of RNA with today's high-throughput sequencing techniques opens the door to methylation studies at nucleotide resolution on a transcriptome-wide scale. Below we describe a protocol for the transcriptome-wide analysis of total or fractionated poly(A)RNA in cells and tissues. Although the nature of the bisulfite sequencing protocol makes it comparably easy to translate from a low to a high-throughput approach, several critical points require attention before starting such a project. We describe a step-by-step protocol for planning and performing the experiment and analyzing the data.

Keywords: 5-Methylcytosine; Bisulfite sequencing; High throughput; Next-generation sequencing; RNA methylation; Transcriptome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / chemistry*
  • Computational Biology / methods
  • Gene Expression Profiling
  • Gene Library
  • High-Throughput Nucleotide Sequencing*
  • Methylation
  • RNA / chemistry*
  • RNA / genetics*
  • RNA / isolation & purification
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification
  • Transcriptome*

Substances

  • RNA, Messenger
  • RNA
  • 5-Methylcytosine