title: GSM4495523: SKO_D60_C3_HBS_seq; Mus musculus; Bisulfite-Seq
library_construction_protocol: Cells were digested using Cell Lysis Solution (Qiagen, Cat. 1045723) and proteins were removed using Protein Precipitation Solution (Qiagen, Cat. 1045701). Genome DNA was precipitated by isopropyl alcohol and cleaned by wash with 75% ethanol. A total amount of 10 μg genomic DNA was mixed with 25 ng lambda and sonicated to 200-bp fragments with Covaris S2 (AB). After purification (PureLink PCR Purification Kit, Invitrogen), DNA fragments were then subjected to end repair with the end repair enzyme mix (NEB), dA tailing using Klenow 3′-5′ exo- (NEB) with purification at each step. Ligation to hairpin adapter (P-GATCGGATGAGCAAGTGAAGCTCATCCGATCT) and Illumina TruSeq adapters were performed using T4 DNA ligase (NEB) overnight. Bisulfite conversion was performed using the EpiTect Bisulphite Kit (Qiagen). After bisulfite conversion, the single-stranded uracil-containing DNA was subjected to 12 cycles of PCR reaction with Illumina TruSeq PCR primers (with specific index) and 2.5 U Pfu TurboCx Hotstart DNA polymerase (Agilent) to recover enough DNA for sequencing. Libraries were sequenced using Illumina HiSeq X10.
identifiers.namespace: GEO
identifiers.id: GSM4495523