GSM9290447: H661-H3K36me2-R2; Homo sapiens; ChIP-Seq
For DPPA2, DPPA4, KDM2A and SUZ12 ChIPs 4x10^6 NCI-H661 cells were utilised per ChIP and underwent double-crosslinking prior to chromatin isolation. All other ChIPs against histone modifications (H3K4me3, H3K4me1, H3K27ac, H3K27me3, H2AK119ub, H3K9me3, H3K36me3, H3K36me2) utilised 0.5x10^6 NCI-H661 or 1x10^6 NCI-H1299 cells per ChIP and underwent single-crosslinking. For double-crosslinking, cells were harvested, washed once with PBS, and resuspended in 10mL of PBS with 5mM EDTA on ice. A fresh stock of 0.24M disuccinimidyl glutarate (ThermoFisher #20593) was reconstituted in DMSO and added to the suspension of cells to reach a final concentration of 2mM, which were then incubated at room temperature whilst rocking for 45 mins to crosslink cells. Cells were pelleted at 3200xg for 5 mins and resuspended in 9.375mL of PBS with 5mM EDTA. Cells undergoing only single-crosslinking were resuspended in 9.375mL PBS at this point. 625uL of fresh 16% formaldehyde (methanol-free) (ThermoFisher, #28908) were added to each cell suspension to reach a final concentration of 1% and then incubated on a rocker for 12.5 mins at room temperature to crosslink cells. Crosslinkers were then quenched by supplementing cell suspensions with 1M ice-cold glycine to reach a final concentration of 125mM and rocked for a further 5 mins at room temperature. Crosslinked cells were then pelleted at 3200xg for 5mins at 4C, washed with 1mL ice-cold PBS with 5mM EDTA and protease inhibitors, and then immediately snap frozen with dry ice and stored at -80C until ChIPs were performed. Crosslinked cell pellets were washed with 1mL ice-cold NP buffer (10mM Tris-HCl, 1M sorbitol, 50mM NaCl, 5mM MgCl2, 1mM CaCl2, 0.1% IGEPAL CA-630, 0.1% Tween-20 in H2O freshly supplemented with 0.385μM beta-mercaptoethanol, 1.82mM spermidine, 1x protease inhibitor tablet) twice to isolate nuclei, with nuclei spun at 2000xg for 5 mins at 4C each time for pelleting. Nuclei were resuspended in 900uL ChIP buffer (20mM Tris-HCl, 2mM EDTA, 150mM NaCl, 0.6% SDS, 1% Triton-X in H2O freshly supplemented with 0.1μM PMSF, 1x protease inhibitor tablet), transferred to a milliTUBE AFA Snap Fibre sonication vial (Covaris, #520135) and left to incubate on ice for 1hr. Chromatin was then  sheared to ~200bp fragments using the ME220 sonicator (Covaris) (4C, 75% power, 15% duty factor, 1000 cycles) for either 30 mins (double-crosslinked) or 20 mins (single-crosslinked). Debris were pelleted at 12,000xg for 15 mins at 4C, and the supernatant containing sheared chromatin was transferred to a new tube. Sheared chromatin was regularly checked through gel electrophoresis to ensure predominantly mononucleosomal fragments. For double-crosslinked ChIPs Protein-G dynabeads were used, and for single-crosslinked ChIPs Protein-A dynabeads were used. 50uL of beads were washed twice in 1mL of low-salt wash buffer (20mM Tris-HCl, 2mM EDTA, 150mM NaCl, 0.1% SDS, 1% Triton-X in H2O) and resuspended in 100uL of low-salt buffer. To pre-clear chromatin, 20uL of washed beads were added to 180uL sheared chromatin and then incubated at 4C for 5 hours on a rotator, followed by transfer of the supernatant to a new tube. To conjugate antibodies to beads, 0.5-4μg of antibodies were diluted to 320uL with ChIP dilution buffer (100mM NaCl, 0.02% sodium azide, 100mM Tris-HCl, 5mM EDTA, 5% Triton-X in H2O supplemented with 1x protease inhibitor tablet) and were left to conjugate with 80uL of washed beads at 4C for 5 hours whilst rotating (IgG controls (0.5-4ug Mouse IgG (Invitrogen, #31903), 0.5-4ug Rabbit IgG (Invitrogen, #012-6102)) or antibodies (4ug DPPA2 (Sigma, #MAB4356), 4ug DPPA4 (abcepta, #AP1438a, abcepta, #AP21202c, abcam, #ab154642), 4ug KDM2A (Invitrogen, #PA5-40421), 1ug SUZ12 (CST, #3737), 0.5ug H3K4me3 (CST, #9751), 0.5ug H3K27me3 (CST, #9733), 0.5ug H2AK119ub (CST, #8240), 0.5ug H3K4me1 (CST, #5326T), 1ug H3K27ac (abcam, #ab4729), 1ug H3K9me3 (abcam, #ab8898), 0.5ug H3K36me2 (abcam, #ab9049), 0.5ug H3K36me3 (CST, #4909)). Antibody-bead conjugates were washed with 1mL of ChIP dilution buffer and subsequently resuspended with 180uL of pre-cleared chromatin and left to incubate overnight whilst rotating at 4C, 9uL of the remaining pre-cleared chromatin was set aside as an input control. Beads were then washed three times with 200uL low-salt wash buffer, three times with 200uL high-salt wash buffer (20mM Tris-HCl, 2mM EDTA, 500mM NaCl, 0.1% SDS, 1% Triton-X in H2O), two times with 200uL LiCl wash buffer (250mM LiCl, 1% sodium deoxycholate, 10mM Tris-HCl, 1mM EDTA, 1% IGEPAL CA-630) and once with 200uL 10mM Tris-HCl pH7.5. Beads as well as input controls were then resuspended/diluted in 50uL ChIP elution buffer (1% SDS, 0.1M NaHCO3 in H2O). Samples were subject to a RNAse A digest (5uL 20mg/mL NEB) for 1hr at 37C, then a Proteinase K digest (5uL 800U/mL NEB) for 1hr at 37C and crosslinks were reversed overnight by incubation at 65C on a thermomixer (1200RPM). DNA was then enriched from eluates using a 1.8X bead cleanup (SergiLabs) and eluted in 30uL H2O for downstream qPCR analyses and DNA library preparation. ChIP 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 NextSeq2000 platform in a single-end configurations with 100bp read lengths.