GSM5990196: M-2616_SK060_WT_10W_crx_WGBS; Mus musculus; Bisulfite-Seq
Whole-genome sequencing libraries were generated from 700 to 1000 ng of genomic DNA spiked with 0.1% (w/w) unmethylated λ DNA (Promega) previously fragmented to 300–400 bp peak sizes using the Covaris focused-ultrasonicator E210. Fragment size was controlled on a Bioanalyzer DNA 1000 Chip (Agilent). The KAPA High Throughput Library Preparation Kit (KAPA Biosystems) 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 KAPA Biosystems' recommendations. The cleaned-up ligation product was then analyzed on a Bioanalyzer High Sensitivity DNA Chip (Agilent) and quantified by PicoGreen (Life Technologies). Samples were then bisulfite converted using the Epitect Fast DNA Bisulfite Kit (Qiagen) according to the manufacturer's protocol. Bisulfite-converted DNA was quantified using OliGreen (Life Technologies) and, based on quantity, amplified by 9–12 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.