GSM4508688: SKN3_WGBS; Homo sapiens; Bisulfite-Seq
DNA was extracted from flash frozen cell pelets of about 5 million cells using DNeasy Blood & Tissue Kit from QIAGEN Cat. #69508. Libraries were generated from 1000 ng of genomic DNA spiked with 0.1% (w/w) unmethylated λ DNA (Roche Diagnostics) and fragmented to 300–400 bp peak sizes using the Covaris focused-ultrasonicator E210. Fragment size was controlled on a Bioanalyzer High Sensitivity DNA Chip (Agilent) and NxSeq AmpFREE Low DNA Library Kit (Lucigen) was applied. End repair of the generated dsDNA with 3' or 5' overhangs, adenylation of 3' ends, adaptor ligation, and clean-up steps were carried out as per Lucigen's recommendations. The cleaned-up ligation product was then analyzed on a Bioanalyzer High Sensitivity DNA Chip (Agilent). Samples were then bisulfite converted using the EZ-DNA Methylation Gold Kit (Zymo Research) according to the manufacturer's protocol. DNA was amplified by 9 cycles of PCR using the Kapa Hifi Uracil + DNA polymerase (KAPA Biosystems) according to the manufacturer's protocol. The amplified libraries were purified using Ampure XP Beads (Beckman Coulter), validated on Bioanalyzer High Sensitivity DNA Chips, and quantified by PicoGreen.
GEO Accession
GSM4508688