title: GSM2692017: KO-biological replicate 1-technical replicate 2; Mus musculus; Bisulfite-Seq
library_construction_protocol: Total RNAs were isolated by Trizol reagent. Poly-A tailed RNAs were enriched by GenElute™ mRNA Miniprep Kit (MRN70-1KT, Sigma). The mRNAs were treated with TURBO DNase, purified by a RNeasy MinElute Cleanup Kit (QIAGEN). Treated mRNAs with 0.5% unmethylated RNA controls from lambda genome were bisulfite converted using the EZ RNA Methylation kit (R5001, Zymo Research). Briefly, 500 ng mRNA was converted using three cycles of 10-min denaturation at 70℃ followed by 45min at 54℃. RNA separation from bisulfite solution, desulfonation, and purification were performed following the standard protocol of the kit. Strand-specific libraries were constructed following the “TruSeq stranded mRNA sample preparation guide” from illumina for 150PE sequencing using Hiseq3000.