GSM9290083: H661-shD4-CTRL-PCGF1-Ms-1; Homo sapiens; OTHER
NCI-H661 or NCI-H1299 cells were harvested and counted after staining cells with 0.4% Trypan blue where 500,000 live cells were used per CUT&Run. Cells were washed with ice cold cell wash buffer (150mM NaCl, 20mM HEPES in H2O) and kept on ice. 11uL Concanavalin A beads (Epicypher) were washed twice with 100uL cold bead activation buffer (20mM HEPES, 50uL KCl, 1mM CaCl2, 1mM MnCl2 in H2O) and then resuspended in 11uL of the same buffer on ice. Cells were washed twice with NE buffer (20mM HEPES, 10mM KCl, 0.1% Triton X, 20% glycerol in H2O) to release nuclei with gentle pipetting and 300xg spins at 4C in between for 5 mins each. Nuclei were resuspended in 100uL of cold cell wash buffer and then incubated with 10uL of washed beads to allow for nuclei-bead adsorption for 10 mins at room temperature. Beads were then resuspended in 50uL of cold antibody buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 0.05% digitonin, 2mM EDTA in H2O) followed by the addition of 1ug of IgG controls (Mouse IgG (Invitrogen, #31903), Rabbit IgG (Invitrogen, #012-6102)) or antibodies (DPPA2 (Sigma, #MAB4356), DPPA4 (abcepta, #AP1438a, abcepta, #AP21202c, abcam, #ab154642), V5-tag (Invitrogen #46-0705), Flag-tag (Merck #F1084 (monoclonal), Invitrogen #740001 (superclonal), RING1B (CST #5694), PCGF1 (Santa Cruz Biotechnology #sc515371), MTF2 (Proteintech #16208-1-AP), EP300 (Aviva Systems Biology (#OAAF01891), SOX2 (abcam #97959)) (or 0.5ug for the H3K4me3 (CST, #9751) positive control) and incubation overnight at 4C on a nutator. Bead complexes were then washed twice with 200uL cold antibody buffer (same as above, but without EDTA) and then resuspended in 50uL of the same buffer. 1.5uL of Protein-A/G-MNase (CST) was added to each bead suspension, pipette mixed and incubated at room temperature for 10 mins. Beads were washed two times with cold 250uL of antibody buffer and resuspended in 50uL of the same buffer. 1uL of 100mM CaCl2 was added to each bead suspension to activate the bound MNase followed by incubation at 4C for 1hr on a thermalcycler to facilitate chromatin digestion. 38uL of stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/mL RNase A, 50ug/mL glycogen in H2O) containing 250pg of E.coli normalisation spike-in DNA (CST) was added to each suspension to inhibit MNase, degrade RNA and release digested chromatin into the supernatant by incubation at 37C for 10 mins in a thermalcycler. The supernatant containing digested chromatin was transferred to a new tube and treated with 1uL 20mg/mL proteinase K for 1hr at 37C. DNA was purified using a 1.8X bead cleanup (SergiLabs) and eluted in 30uL of H20 for DNA library preparation. CUT&Run libraries were prepared using the NEBNext UltraII DNA Library Preparation Kit (NEB) according to the manufacturer's instruction. During library preparation the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-4) were used to enable library multiplexing. Individual and pooled libraries were quantified and quality controlled using the dsDNA high sensitivity (HS) Qubit assay (Invitrogen) on the Qubit 3.0 fluorometer (Invitrogen) and HSD1000 High Sensitivity DNA kit (Agilent) on the TapeStation 4150 (Agilent). Libraries were pooled and sequenced on the Illumina NextSeq2000 platform in a paired-end configurations with 100bp read lengths.