GSM5961450: MiaPaCa-2_KD2; Homo sapiens; Bisulfite-Seq
About 1 X 10^7 cells were lysed with 900µl TRI reagent, and the total RNA was extracted with Direct-zol RNA miniPrep Plus (Zymo, Irvine, CA)  according to manufacturer's instruction. Average 30-40 µg total RNA was proceed to mRNA enrichment, which were done by oligo (dT)25 magnetic beads from Dynabeads mRNA DIRECT purification kit (Thermo Fisher Scientific, Waltham, MA). Then, the enriched mRNA samples were subjected to bisulfite conversion using EZ RNA methylation kit (Zymo, Irvine, CA) according to manufacturer's instruction. With one adjustment that the incubation step was modified into three cycles of 70℃ 10 minutes and 64℃ for 45 minutes followed by Amort et al. method. About 20ng of bisulfite-treated mRNA was used to generate sequencing library. Libraries were built with NEBNext Ultra Ⅱ Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) arrording to manufacturer's instruction. The fragmentation step was omitted due to previous bisulfite treatment had already resulted in fragmentation of RNA sequences to 200-500nt.