Transcriptome-Wide Detection of 5-Methylcytosine by Bisulfite Sequencing
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.
Key wordsBisulfite sequencing RNA methylation 5-Methylcytosine High throughput Transcriptome Next-generation sequencing
Funding in A.L’s lab is provided by the Austrian Science Fund (FWF): P27024-BBL.
- 1.Grosjean H (2009) Nucleic acids are not boring long polymers of only four types of nucleotides: a guided tour. In: Grosjean H (ed) DNA and RNA modification enzymes: structure, mechanism, function and evolution. LandesBioscience, AustinGoogle Scholar
- 9.Rieder D, Amort T, Kugler E, Lusser A, Trajanoski Z (2015) meRanTK: methylated RNA analysis ToolKit. Bioinformatics. doi: 10.1093/bioinformatics/btv647