GSM9848419: 41978_KH_IgG_BX_rep1-1; Homo sapiens; ChIP-Seq
Cells were crosslinked in methanol-free formaldehyde (final 1% concentration in IMDM media) at 25 °C for 10 min. 1 mole of formaldehyde was quenched with 3 mole extra Tris (pH 8.0) at 25 °C for 5 min. Fixed cells were washed with PBS and stored at -80 oC. 25 million (M) formaldehyde crosslinked K562 were lysed by incubating with 1 mL Lysis Buffer F (20 mM Tris, pH 8.0, 85 mM KCl, 0.5% NP-40, 0.5% Triton X-100) for 20 min at 4°C, then with 1 mL RIPA Buffer M (1x PBS 1.0% NP40, 0.5% NaDeoxycholate, 0.1% SDS) for 20 min at 4°C. Nuclei (25 M) were incubated in 500 ul Benzonase Digestion Buffer (10 mM Tris, pH 8, 10 mM NaCl, 2 mM MgCl2) at a Benzonase concentration of 100 U/ml for 10 min at 25 °C. Reactions were quenched by adding 500 ul Benzonase Quench Buffer (10 mM Tris, pH 8, 800 mM NaCl, 20 mM EDTA), The quenched reactions were centrifuged at 14,000 rpm for 10 minutes to collect Benzonase-solubilized chromatin in the supernatant. Nuclei pellets were sonicated in a Bioruptor PICO model sonicator (Diagenode) for 8 cycles (30-on/ 30-off). Sonicated material was centrifuged at 13.2K rpm for 15 minutes using a Microfuge (Eppendorf 5415R). Solubilized chromatin supernatant was collected. Benzonase solubilized and sonication chromatin were collected for incubation with pAG Mag Sepharose ™ (Cytiva™) preloaded with 3 ug of antibody overnight. Following antibodies were used according to the manufacturer's instructions to prepare ChIP-exo libraries: SMC3 (ab9263, Sigma), P300 (HPA003128, Atlas), NFIA (HPA008884, Atlas), WDR5 (HPA047182, Atlas), GABPA (HPA003258, Atlas), SP1 (HPA001853, Atlas), RAD21(HPA020044, Atlas), GTFIIB (HPA061626, Atlas), P300(A300-358A, Bethyl labs), RbBP5 (A300-109A, Bethyl labs), and CTCF (07-729, Millipore). TBP (Pugh lab). The immunoprecipitated chromatin was then processed through Lambda exonuclease digestion, first adapter ligation, cross-link reversal, second adapter ligation, and PCR for final high-throughput sequencing. Illumina or Element library prep