GSM7426510: HSSY, sgPCGF1, input, R3; Homo sapiens; ChIP-Seq
The experiment was performed in biological triplicate. Drosophila cell line SG-4 was used as the spike-in cell line. HSSY-II were transduced with either the empty vector plasmid or with sgRNA targeting the PCGF1 gene. The cells were cultured for 10 days and after that 10*10^6 cells were harvested; 2*10^6 SG-4 cells were mixed with the harvested cells. The cells were washed with ice-cold lysis buffer (10 mM Tris-HCl at pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 5 mM sodium butyrate, 5 mM N-ethylmaleimide) to extract the nuclei. The nuclei were then digested using 100U of MNase (Fermentas EN0191) at 37°C for 5 minutes in MNase digestion buffer (10 mM Tris-HCl at pH8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25 M sucrose, 3 mM CaCl2, 10 mM sodium butyrate, 10 mM N-ethylmaleimide, 1× PIC (Roche) followed by an addition of 4 mM EDTA to stop the digestion. All the buffers were supplemented with protease inhibitor and NEM (inhibitor of de-ubiquitinase enzymes). After centrifugation, the supernatant was retained and incubated overnight at 4°C with 5 μL of anti-H2AK119ub1 (Cell Signaling Technology, D27C4). Next, 30ul protein A/G magnetic beads (Thermofisher) were added for the pull down and incubated for 2h at 4°C. To elute the DNA, beads were incubated in 1% SDS, 0.1M NaHCO3 for 30 minutes at 24°C. DNA was purified using ZymoResearch ChIP DNA clean and concentrator kit. DNA fragments obtained after ChIP were quantified using Qubit dsDNA HS Assay Kit (Invitrogen). 5ng of DNA was used for libraries preparation using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S), SPRIselect beads (Beckman Coulter, #B23317) and NEBNext Multiplex Oligos for Illumina (NEB, Set 1 #E7335S, Set 2 #E7500S). ChIP libraries were done following NEB's guidelines (NEB, E7645S)