5-Methylcytosine Analysis by RNA-BisSeq

Methods Mol Biol. 2019:1870:237-248. doi: 10.1007/978-1-4939-8808-2_18.

Abstract

5-Methylcytosine (m5C) is a posttranscriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. Many known or novel m5C sites have been validated by using advanced high-throughput techniques combined with next-generation sequencing (NGS), especially RNA bisulfite sequencing (RNA-BisSeq). Here we introduce an optimized RNA-BisSeq method by using ACT random hexamers to prime the reverse transcription of bisulfite-treated RNA samples to detect the m5C sites.

Keywords: ACT random hexamers; Sodium bisulfite; UHPLC; cDNA libraries; mRNA.

MeSH terms

  • 5-Methylcytosine* / metabolism
  • Chromatography, High Pressure Liquid
  • Computational Biology / methods*
  • Databases, Nucleic Acid
  • High-Throughput Nucleotide Sequencing* / methods
  • Mass Spectrometry
  • Methylation
  • RNA / chemistry
  • RNA / genetics*
  • RNA / metabolism
  • RNA Processing, Post-Transcriptional*
  • RNA, Messenger / genetics
  • Software

Substances

  • RNA, Messenger
  • RNA
  • 5-Methylcytosine