title: GSM5899652: ChIP-seq Pol2Ser2 Control; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation was performed as previously described [Izumi, K. et al. Nat Genet, 2015, doi:10.1038/ng.3229]. In Brief, ~ 8 × 106 human cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by an additional 5 min with glycine in PBS added at a final concentration of 125 mM. Fixed cells were lysed in LB1 (50 mM HEPES-KOH (pH 7.4); 140 mM NaCl; 1 mM EDTA; 10% glycerol; 0.5% NP-40; 0.25% Triton X-100; 10 mM dithiothreitol; 1 mM PMSF) on ice. The lysate was wash with LB2 (20 mM Tris-HCl (pH 8.0); 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1mM PMSF) and LB3 (20 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% SDS; 1 × cOmplete protease inhibitor cocktail (Roche)) on ice. The lysate was resuspended in LB3 and sonicated using Branson Sonifier 250D (Branson) for chromatin shearing (12 sec with amplitude setting at 17% of the maximum amplitude, 6 times). In addition, lysate containing fragmented chromatin was also prepared from ~ 2 × 106 C2C12 mouse cells with the same procedures. Human cell lysate and mouse cell lysate (as spike-in internal control) were combined (approximately 4:1 ratio) and incubated with protein A or G Dynabeads (Thermo Fisher Scientific) conjugated with antibodies for 14 h at  4℃. The beads were then washed 5 times with cold RIPA wash buffer (50 mM HEPES-KOH (pH 7.4); 500 mM LiCl, 1 mM EDTA; 0.5% sodium deoxycholate; 1% NP-40) and once with cold TE50 (50 mM Tris-HCl (pH 8.0); 10 mM EDTA). Material captured on the beads was eluted by TE50 containing 1% SDS. The eluted material and input were incubated for 6 h at 65℃ to reverse crosslinks and treated with 100 ng RNaseA (Roche) for 1 h at 37℃, followed by treatment  with 100 ng Proteinase K (Merck) overnight at 50℃. The input and ChIP DNA were then purified by PCR purification kit (Qiagen) following manufacturer's instructions and further sheared to an average size of approximately 150 bp using Covaris S2 focused-ultrasnicator (settings: Duty Cycle, 10%; Intensity, 5; Cycle per Burst, 100; Duration, 300 sec). The sheared DNA was end-repaired, ligated to sequencing adaptors, amplified and size-selected using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and Agencourt AMPure XP (Beckman Coulter) following manufacturer's instructions. Sequencing libraries were made using the NEBNext ChIP-Seq Library Prep Master Mix Set of Illumina (New England Biolabs).