GSM2196128: KO ESC #1; Mus musculus; Bisulfite-Seq
Genomic DNA extraction was performed with QIAamp DNA mini kits (Qiagen) following manufacturer's instructions. 50 ng of genomic DNA was digested in 18 µl of MspI digestion master mix containing 10 units of MspI (Thermo Scientific) and 1.8 µl 10x Buffer Tango (Thermo Scientific) for 3 hours at 37 ℃. After 20 minutes of inactivation at 80 ℃, the digested DNA was filled-in and tailed with an extra A to the 3’ end by adding 2 µl of end-preparation master mix containing 5 units of Klenow Fragment (exo-; Thermo Scientific), 1.8 µl of 10x Buffer Tango, 0.4 mM dATP, and 0.04 mM each of dGTP and dCTP (Thermo Scientific), followed by 40 minutes of incubation at 37 ℃ and 15 minutes of inactivation at 75 ℃. Adaptor ligation was then performed by adding 5 µl of ligation master mix, which contains 30 Weiss Units of T4 DNA Ligase (HC, Thermo Scientific), 0.5 µl of 10x Buffer Tango, 5mM ATP (Thermo Scientific), and 150 nM of methylated custom adaptor (Forward: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’, Reverse: 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’, where * indicates phosphorothioate bond). After three incubation steps (16 ℃ for 30 min, 4 ℃ overnight, and 65 ℃ for 20 min), the reaction mix was subjected to EpiTect Fast Bisulfite Conversion Kit (QIAGEN) following manufacturer’s low-concentration protocol. The bisulfite converted DNA was immediately subjected to PCR amplification using KAPA HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems) and NEBNext Primers for Illumina (New England Biolabs). After amplification, final libraries were obtained by size selection of 150-500 bp DNA fragments using SPRIselect beads (Beckman Coulter).