Project description:Total RNA was isolated from mouse lung harvested at different stages using TRIzol® Reagent (Ambion). mRNA was extracted with using Dynabeads® mRNA Purification Kit (Ambion) and subjected to TURBO™ DNase (Invitrogen) treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA-BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 (Dhfr mRNA serves as methylation conversion control) was fragmented to ~100-nt fragments for 1 min at 90°C in 10× RNA Fragmentation Reagent (Ambion), then stopped by 10× RNA stop solution (Ambion), and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution (pH 5.1), which is a 100:1 mixture of 40% sodium bisulfite (Sigma) and 600 µM hydroquinone (Sigma) and subjected to heat incubation at 75°C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns (PALL Corporation) with centrifugation. After washed with nuclease-free water and centrifuged for five times, the RNA was finally disolved in 75 μl nuclease-free water and then desulfonated by incubation with an equal volume of 1 M Tris-HCl (pH 9.0) at 75 °C for 1 h. After ethanol precipitation, the RNA was resuspended in 11 µl of RNase-free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase (Invitrogen) and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA-Seq Kit (KAPA) according to the manufacturer’s instructions.Libraries were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 125 bp.
