GSM9193230: ChIP-nexus on wildtype mESC R1 cells, CRISPR side-by-side control, replicate 1; Mus musculus; ChIP-Seq
ChIP-nexus experiments were performed using 10 million cells per ChIP. The cells were first washed once with PBS, crosslinked using 1% formaldehyde, and quenched after 10 minutes using 125 mM glycine. Fixed cells were then washed twice more with ice cold PBS and resuspended in chilled lysis buffer (15 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate and 0.1% SDS). After a 10 minute incubation on ice, a Bioruptor Pico (Diagenode) was used to sonicate the chromatin with five or six cycles, 30 seconds on/off. The commercially available Sox2 (Active Motif, 39843) antibody was used for these experiments. For each ChIP, 10 µg of antibody was conjugated to 50 µL of Protein A or G dynabeads (Invitrogen). Following the ChIP, ChIP-nexus library preparation was performed including end repair, dA-tailing, adapter ligation, barcode extension, and lambda exonuclease digestion. Addition of the ChIP-nexus adapter mix containing four fixed barcodes and PCR library amplification was performed directly after circularization of the purified DNA fragments (without addition of the oligo and BamHI digestion).