GSM4593062: WT_ESC_CBX7_ChIP_group1; Mus musculus; ChIP-Seq
Ten to twenty million harvested cells were crosslinked in 1% formaldehyde-PBS for 10 minutes at room temperature. Crosslinking was quenched with glycine, and cells were pelleted by centrifugation at 4°C and washed once in cold PBS. Cells were resuspended in cold lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA pH 8.0, 1% SDS, PIC, 1mM DTT) and incubated for 10 minutes on ice prior to sonication using a QSonica Q800R3 Sonicator. Insoluble chromatin was pelleted by centrifugation, and supernatant was diluted 1:10 with IP Dilution Buffer (20mM Tris-HCl pH 8.1, 150mM NaCl, 2mM EDTA pH 8.0, 0.1% Triton X-100, PIC, 1mM DTT). Sonicated Drosophila chromatin was added at a known ratio to experimental chromatin to serve as a spike-in as previously described (Egan et al., 2016). Triton X-100 was added to a final concentration of 1%, input was collected and diluted to 200mL, and chromatin was added to Protein A Dyanbeads (Invitrogen) that had been previously incubated with experimental and spike-in antibodies. Samples were rotated at 4°C overnight. Beads were washed with IP Dilution Buffer with 1% Triton X-100, followed by Wash Buffer A (50mM HEPES pH 7.9, 500mM NaCl, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, PIC, DTT), followed by Wash Buffer B (20mM Tris-HCl pH 8.0, 250mM LiCl, 1mM EDTA pH 8.0, 0.1% Na-deoxycholate, 1% Igepal, PIC, DTT), followed by a quick TE wash. Beads were incubated at 37°C in 200mL Extraction Buffer (1% SDS, 0.1M NaHCO3) for 15 minutes with shaking. Supernatant was collected, and 12mL 2M TrisHCl, 2mL RNase A (500mg/mL, Roche), and 12mL 5M NaCl were added to ChIP samples and inputs. Crosslinks were reversed overnight at 65°C, followed by 1 hour incubation at 55°C with Proteinase K. DNA was isolated by phenol:chloroform extraction and ethanol precipitation. Libraries were prepared as described in Bowman et al., 2013 (BMC Genomics). In brief, this protocol includes end repair, A-tailing, adapter ligation, and real-time PCR amplification, with SPRI cleanup of samples between each step.
GEO
GSM4593062