Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.
To ensure decapping and phosphorylation of the 5' end of the RNA, 500?ng of the bisulfite-converted sample was treated with five units of Tobacco Acid Pyrophosphatase (Epicentre Biotechnologies) at 37°C for 45?min followed by phenol/chloroform extraction. The sample was then subjected to T4 Polynucleotide Kinase (New England Biolabs) treatment and subsequent phenol/chloroform extraction. A next-generation sequencing library was prepared using the SOLiD™ Whole Transcriptome Analysis kit (Applied Biosystems). Since the sample was already sufficiently fragmented by the bisulfite-conversion reaction, the RNA fragmentation step prior to adaptor hybridization and ligation was not performed. cDNA with an approximate insert length of 50–120?nt were selected by polyacrylamide gel electrophoresis. Beads were prepared, deposited and sequenced on a SOLiD™ Version 3 instrument (Applied Biosystems).