title: GSM3475615: Undifferentiated keratinocytes, replicate 3; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Total RNA of about 4 μg was extracted using Trizol (ThermoFisher) and DNase (Ambion) then Ribo‐Zero (Illumina) treated. The remaining RNA fraction was bisulfite‐treated according to manufacturer's guidelines. Briefly, ribosomal depleted RNA was mixed with 70 μl of 40% sodium bisulfite pH 5.0 and DNA protection buffer (EpiTect Bisulfite Kit, Qiagen). The reaction mixture was incubated for three cycles of 5 min at 70°C followed by 1 h at 60°C and then desalted with Micro Bio‐spin 6 chromatography columns (Bio‐Rad). RNA was desulphonated by adding an equal volume of 1 M Tris (pH 9.0) to the reaction mixture and incubated for 1 h at 37°C, followed by ethanol precipitation. Bisulfite converted RNA was treated with T4 PNK (New England Biolabs) to repair both 5' and 3' ends for library preparation. Repaired RNA quality and concentration was measured on a Bioanalyzer 2100 RNA nano‐chip (Agilent).  About 100 ng of RNA was used to generate Bisulfite‐seq libraries. Libraries were performed according to TruSeq Small RNA preparation kit (Illumina). RNA adapters were then ligated, reverse‐transcribed followed by 18‐cycle PCR amplification before sequenced. 
attributes.tag: GEO Accession
attributes.value: GSM3475615