title: GSM6149733: HEK293T_POLH_1; Homo sapiens; ChIP-Seq
library_construction_protocol: Briefly, 2×107 cells were cross-linked by adding formaldehyde dropwise directly to the media for a final concentration of 1% and rotating gently at room temperature for 10 min. The reaction was quenched by adding 2.5 M glycine to the media for a final concentration of 125 mM and incubated with shaking for 5 min at room temperature. After washing with 1× PBS three times, the cells were resuspended in PBS. After centrifugation, the cell pellet was resuspended in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS and protease inhibitor cocktail) at 4 °C for 30 min with rotation. Chromatin was sheared using a Covaris S220 sonicator at 4°C for 10 min with a peak incident power of 140, a duty cycle of 10%, and 200 cycles per burst. After centrifugation at 16,000g for 15 min, the supernatant was incubated with pre-blocked anti-FLAG M2 affinity gel (Sigma) on a rotator at 4°C overnight. After washing, DNA was eluted from the beads with 120 µL 100 mM NaHCO3 and 1% SDS at 68 °C for 2 h. Cross-links were subsequently reversed by adding 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubated with shaking at 65°C overnight. After reverse cross-links, proteins in the resulting DNA samples were removed with 2 µL proteinase K (20 mg/mL) and incubated while shaking at 60°C for 2 h. Finally, the DNA was purified using Cycle Pure Kit (omega). Purified DNA was quantified and verified on an Agilent 2100 Bioanalyzer. The library was then constructed using NEBNext Ultra DNA Library Prep Kit (NEB, E7103S) following the manufacturer's instructions. Subsequently, the purified DNA libraries were assessed using an Agilent 2100 Bioanalyzer and multiplexed for sequencing on a HiSeq4000 system (Illumina).