GSM5706852: HU090_B24_K562_RAD21-mAC_4_IAA_G1_antiMCM5_ChIP-input; Homo sapiens; ChIP-Seq
Collected cells were fixed by 1% formaldehyde (Sigma, F1635, fresh-made) for 10 min at 25oC and quenched by 125 mM glycine (VWR-amerasco 0167) for 5 min. Thoroughly washed by PBS, cells were lysed on ice for 15 min with ice-cold NP40 lysis buffer (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% NP40) supplied with protease inhibitors (PIs) (Bimake, B14012). The nuclei fraction was isolated, washed by PBS/1 mM EDTA, and then lysed with glycerol buffer (20 mM Tris-HCl, pH 8.0; 75 mM NaCl; 0.5 mM EDTA; 0.85 mM DTT; 50% glycerol) and nuclei lysis buffer (10 mM HEPES, pH 7.6; 1 mM DTT; 7.5 mM MgCl2; 0.2 mM EDTA; 0.3 M NaCl; 1 M urea; 1% NP40). The chromatin was pelleted by centrifuge, washed twice by PBS/1 mM EDTA, and eventually suspended in sonication buffer (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2 mM EDTA; 0.1% SDS; 1% Triton X-100; 4 mM CaCl2) with PIs. Chromatin fractions were then treated with 50 U MNase (NEB, M2047S) for 10-12 min at 37oC, and the digestion was quenched by adding 5 mM EGTA and 5 mM EDTA. After that, pre-digested chromatin was sonicated by a Biorupter (Energy: High; On: 30 s; Off: 60 s; 15 cycles; 4oC). 30 mL of soluble chromatin fraction was kept as input and the rest was subjected to ChIP analysis. For ChIP-seq, an antibody against  MCM5 (1:50; Abcam, ab75975) was incubated with the soluble chromatin fraction for 2 hours at 4oC. 40 mL protein G dynabeads (Invitrogen, 10003D) were then added overnight. Next, beads were sequentially washed by high-salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.0) for twice, LiCl buffer (0.25 M LiCl; 1% NP-40; 10 mM Tris-HCl; pH 8.0, 1 mM EDTA) for once, and TE buffer for three times. Chromatin bound on beads was eluted twice with elution buffer (20 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS) at 65 oC, each for 15 min. Eluate fraction was treated with RNase A (Thermo Fisher, EN0531), Proteinase K (Invitrogen, 25530015), and de-crosslinked at 65 oC overnight. Purified DNA was end-repaired, tailed with poly-dC, tagged with biotin, captured by Streptavidin C1 beads (Invitrogen, 65002), and ligated with an adapter. The beads-bound DNA was thoroughly washed, tagged with Illumina sequences, and sequenced with Hi-seq (2×150 bp).