title: GSM8316456: Old_HSC_CD150L_RRBS_rep4; Mus musculus; Bisulfite-Seq
library_construction_protocol: FACS sorted HSCs were lysed at 50°C for 3 hours and 75°C for 30 min in a 5 μl lysis buffer [20 mM tris-EDTA (Sigma catalog # T9285-100ML), 20 mM KCl (Thermo Scientific, catalog # AM9640G), 0.3% Triton X-100 (Sigma catalog # 93443-100ML), and protease (1 mg/ml; Qiagen catalog # 19155)]. The lysed cells were then subject to Msp I digestion at 37°C for 3 hours and 80°C for 20 min. Following enzymatic digestion, end-repair mix [1 μl Klenow frag exo− (5 U/μl), 0.2 μl of 10× TangoBuf, and 0.8 μl of dNTP mix] was added and incubated at 37°C for 40 min and 75°C for 15 min. For adaptor ligation, the reaction was incubated at 16°C for 30 min, 4°C overnight, and 65°C for 20 min after adding the ligation mix [2.25 μl of H2O, 1 μl of T4 ligase (30 U/μl; Thermo Scientific, catalog # EL0013 ), 0.5 μl of 100 mM ATP (Thermo Scientific, catalog # R0441), and 0.75 μM methylated adaptor. Bisulfite conversion reaction was then performed by the EpiTect Bisulfite Kit (Qiagen catalog # 59104) following the manufacturer's instructions. Adapter-tagged BS-DNA was then amplified by 16 cycles using Kapa HiFi Uracil+ ReadyMix (Kapa Biosystems, catalog # KK2801) with NEBNext Multiplex Oligos for Illumina (New England BioLabs catalog # E7500L).