GSM1206696: Het-1 sperm; Mus musculus; Bisulfite-Seq
DNA was extracted in DNA lysis buffer (10 mM Tris-HCl (pH 8.5), 10 mM EDTA, 0.2% SDS, 200 mM NaCl) supplemented with 300 mg/ml proteinase K followed by phenol:chloroform extraction and ethanol precipitation. DNA was digested with MspI (NEB), and then subjected to end repair, and ligation of methylated custom adaptors (Forward: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’, Reverse: 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’, where * indicates phosphorothioate bond) with a NEBNext Ultra DNA Library Prep Kit for Illumina (NEB). Size selection was conducted by purifying the adaptor-ligated DNA twice with 1.5× SPRIselect beads (Beckman Coulter). Size selected libraries were treated with sodium bisulfite using the EpiTect Fast Bisulfite Conversion Kit (QIAGEN). After clean-up, the optimal, minimum PCR cycle numbers required to generate the final libraries were determined by qPCR using KAPA 2G Robust HotStart ReadyMix (KAPA) with 0.3× SYBR I. Final libraries were generated by scaled-up PCR reactions (without SYBR I) using the cycles determined above with barcoded primers from NEBNext Multiplex Oligos kit (NEB), and purified with 1.2× SPRIselect beads.