GSM8526224: C17_RAD21AID_D8,mCherry,MAU2,rep1; Homo sapiens; ChIP-Seq
100 million cells were crosslinked with 1% formaldehyde for 10min. Cells were subsequently quenched with 125mM glycine for 10min and washed twice with cold PBS. Cells were scraped from culture dishes and cell pellets were subsequently lysed in LB1 buffer (50mM Hepes, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100), washed in LB2 buffer (10mM Tris, 200mM NaCl, 1mM EDTA, 0.5mM EGTA), and resuspended in LB3 buffer (10mM Tris, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine) prior sonication. Chromatin was sonicated using Bioruptor Pico (Diagnode) with a setting of 30s on, 30s off for 8 cycles. Fragmented chromatin was then incubated with 6ug of antibodies pre-coupled to Dynabeads protein G beads (Thermofisher) overnight at 4oC. Beads-bound chromatin was then washed 10x with RIPA buffer (50mM Hepes, 500mM LiCl, 1mM EDTA, 1% NP40, 0.7% sodium deoxycholate), once with TBS buffer, and decrosslinked in elution buffer (50mM Tris, 10mM EDTA, 1% SDS) at 65oC for 18h. Eluted DNA was then treated with protease K and RNAse A, and subsequently purified with phenol/chloroform/isoamyl alcohol 25:24:1. Purified DNA was either assessed with qPCR or continued with ChIP-seq NGS sequencing library preparation. Sequencing libraries was constructed using NEBnext Ultra II DNA library prep kit (NEB) following the manufacture's protocol. Briefly, DNA was end-repaired and poly-A tailed, ligated to NEBnext adapters, and digested with USER enzyme. Annealed libraries were then purified with AMPure XP beads (Beckman Coulter) and PCR amplified with indexing primers for 4-12 cycles. Sequencing libraries were checked with Bioanalyzer HS DNA chip (Agilent).