GSM3462641: Spleen_N6 (human_BS); Homo sapiens; Bisulfite-Seq
Total RNA was isolated from tissues or cultured cells using chloroform and isopropanol following the manufacturer's protocol. Polyadenylated RNA was separated from total RNA using either GenElute mRNA miniprepKit (Sigma-Aldrich) or Oligo dT Magnetic Beads (Vazyme). Bisulfite treatment was performed using the EZ RNA methylation kit (Zymo Research) with some modifications. In brief, 1μg of polyadenylated RNA was converted using three different conversion conditions. The quantity of the converted RNA was determined by Qubit. Libraries were then constructed using VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme). In short, 20ng converted RNA was fragmented into 150~200 nt fragments by incubation at 94 °C for 3min in 2× frag/prime buffer. The fragmented RNA was then used for library construction, following the manufacturer's protocol.