RNA m5C analysis in human urothelial carcinoma of the bladder (UCB) and adjacent normal tissues.. RNA m5C analysis in human urothelial carcinoma of the bladder (UCB) and adjacent normal tissues.

36 cases of UCB and 29 adjacent normal bladder tissues (22 pairs) were derived from patients diagnosed with UCB and received radial cystectomy at Sun Yat-sen University Cancer Center (SYSUCC, Guangzhou, China).Total RNA was isolated using TRIzol® Reagent (Ambion). The RNA concentration and quality were determined with NanoDrop, Qubit and agarose gel analysis. The Dynabeads® mRNA Purification Kit (Ambion) was used to enrich the mRNAs, which were used as templates for RNA-BisSeq library construction. For RNA-BisSeq, bisulfite-treated mRNAs were first subjected to reverse transcription with ACT hexamers and Superscript II Reverse Transcriptase (Invitrogen), and the following processes were carried out with KAPA mRNA Stranded mRNA-Seq Kit (KAPA) according to the manufacturer’s instructions. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length. The m5C sites were called using meRanCall from meRanTK (FDR < 0.01). The differential m5C sites were detected and the hypermethylated m5C in UCB samples correlates with oncogene mRNA overexpression in UCBs. Overall design: Examination of RNA m5C changes in UCB patients