GSM7785122: T47D H3K4me3, ChIP, hypoxia, biol rep1; Homo sapiens; ChIP-Seq
Post hypoxia treatment, control and treated cells were washed 2x in PBS and collected (500 rcf for 5 minutes at 4°C) and flash frozen. Prior to each ChIP, antibodies were conjugated overnight at 4°C in ChIP Buffer 1 (25 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 10% glycerol, 0.1% NP-40, 200 uM PMSF, 50 ug/mL BSA) with 50/50 mix of Dynabeads™ Protein A (Thermo Fisher, 10001D) and Dynabeads™ Protein G (Thermo Fisher, 10003D) at a ratio of 6 ug of antibody to 25 uL of beads per sample. Prior to IP, beads were washed 2x with ChIP Buffer 1 and resuspended in 25 uL of ChIP Buffer 1. Frozen cells were washed twice in Buffer N (15 mM Tris Base, 15 mM NaCl, 60 mM KCl, 8.5% sucrose, 5 mM MgCl2, 1 mM CaCl2, 1 mM DTT, 200 uM PMSF, 1x protease inhibitor, 50 ug/mL BSA), then resuspended in 2x cell pellet volume of Buffer N and lysed for 10 minutes on ice in equivalent volume of 2x Lysis Buffer (1x Buffer N, 0.6% NP-40). Nuclei was pelleted (500 rcf for 5 minutes at 4°C) and resuspended in 6x cell pellet volume of Buffer N, then spun through Sucrose Cushion (15 mM Tris Base, 15 mM NaCl, 60mM KCl, 30% sucrose, 5 mM MgCl2, 1 mM CaCl2, 1 mM DTT, 200 uM PMSF, 1x protease inhibitor, 50 ug/mL BSA) at 500 rcf for 12 minutes at 4°C. Nuclei pellet was resuspended in 2x cell pellet volume of Buffer N. 2 uL of nuclei suspension was diluted with 18 uL of 2 M NaCl in triplicate, vortexed, and sonicated with a water bath sonicator (30x cycle of 30 seconds on, 30 seconds off) prior to measuring the chromatin concentration with NanoDrop Spectrophotometer (Thermo Fisher). Concentration of nuclei suspension was then adjusted to 1 ug/uL and separated into 100ug chromatin aliquots. Nuclei suspensions were prewarmed for 2 minutes at 37°C, then 1.52 uL of MNase (Worthington Biochemical Corporation, LS004797) was added for 12 minutes at 37 °C on a ThermoMixer (Eppendorf) set to 900 rpm. Digestion was stopped by adding 1/10 volume of MNase Stop Buffer (100 mM EDTA and 10 mM EGTA) and placing nuclei on ice. 5M NaCl was slowly added to the suspension to a concentration of 0.6 M on the slowest vortex setting to release the chromatin, insoluble nuclei was then pelleted (18,000 rcf, 1 minute, 4 °C), and supernatant was run through hydroxyapatite (HAP) chromatography. 15 uL of soluble chromatin was saved for visualization and the rest of the soluble chromatin was added to 0.07 g of HAP resin (Bio-Rad, #157-0021) rehydrated in HAP Buffer 1 (5 mM NaPO4 pH 7.2, 600 mM NaCl, 1 mM EDTA, 200 uM PMSF) and rotated for 10 minutes at 4 °C. HAP mixture was run through Ultrafree-MC Centrifugal Filter (Millipore, UFC30HV25) at 600 rcf for 30 seconds at 4 °C and 15 uL of flow-through was saved for visualization. Then the column was washed 4x with 200uL HAP Buffer 1, 4x with 200 uL HAP Buffer 2 (5 mM NaPO4 pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 uM PMSF), and eluted 3x with 100 uL of HAP Elution Buffer (500 mM NaPO4 pH 7.2, 100 mM NaCl, 1 mM EDTA, 200 uM PMSF). Flowthrough after each step was saved for visualization. Chromatin concentration was measured again in triplicate with the combined HAP elutions using NanoDrop. Final chromatin concentration was adjusted to 20 ng/uL using ChIP Buffer 1 prior to IP. 15 uL of chromatin was set aside for input control and chromatin was added to beads conjugated with H3K4me3 antibody (Epigentek, A4033). Chromatin and bead mixture was rotated for 10 minutes at 4 °C, washed 2x with ChIP Buffer 2 (25 mM Tris pH 7.5, 5 mM MgCl2, 300 mM KCl, 10% glycerol, 0.1% NP-40, 200 uM PMSF, 50 ug/mL BSA), washed 1x ChIP Buffer 3 (10 mM Tris pH 7.5, 250 mM LiCl, 1 mM EDTA, 0.5% Sodium Deoxycholate, 0.5% NP-40, 200 uM PMSF, 50 ug/mL BSA), and washed 1x with TE buffer, transferring beads to a new tube after each wash step. DNA was finally eluted in 50 uL of ChIP Elution Buffer (50 mM Tris pH 7.5, 1 mM EDTA, 1% SDS) at 600 rpm for 10 minutes at 55 °C on a ThermoMixer. 35 uL of ChIP Elution Buffer was also added to the input control to bring the final volume to 50 uL. To both input and eluted ChIP DNA, 2 uL of 5 M NaCl, 1uL of 500 mM EDTA, and 1 uL of 20 mg/mL Proteinase K was added and incubated for 2 hours at 55 °C and DNA was purified using MinElute PCR Purification Kit (Qiagen, 28004). DNA as eluted in ultrapure H2O and concentration was measured before and after purification using Qubit dsDNA Quantitation, High Sensitivity (Thermo Fisher, Q32851). Finally, HAP input and elutions were visualized using 2% agarose gel in 1x TBE ran at 140 V for 40 minutes to confirm fragment sizes. ChIP DNA was sent to the McGill University and Genome Quebec Innovation Center, where it was further validated via Bioanalyzer, followed by shotgun library preparation, and library sequencing via Illumina NovaSeq6000. H3K4me3 ChIP DNA was sequenced to a depth of about 50 million reads and input samples were sequence to a depth of about 150 million reads.