GSM8084410: HCT116_Cl3_shU1_Baseline_D14_K9me3_R1; Homo sapiens; ChIP-Seq
Cells  were fixed in buffer (1% formaldehyde, 50 mM HEPES-KOH pH 7.6, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) for 10 min at room temp with shaking and then quenched with 125 mM glycine 5 min at room temp. Cells were scraped into cold PBS, washed 2X with cold PBS, flash frozen in liquid N2, and stored at -80°C until use. Thawed pellets were lysed in LB1 (50 mM HEPES-KOH pH 7.6, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, Roche protease inhibitor cocktail) for 20 min with rotation at 4°C and cleared by centrifugation at 300 x g for 5 min at 4°C. Supernatant with intact nuclei was set aside. Cell pellets were lysed again in 4x LB1 (LB1 with 2% NP-40 and 1% Triton X-100) for 20 min. Intact nuclei from this and the saved supernatant were collected by centrifugation at 1,700 x g for 5 min at 4°C, resuspended and washed in LB2 (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, protease cocktail inhibitor) for 10 min with rotation at 4°C, and collected again by centrifugation at 1,700 x g for 5 min at 4°C. Nuclei were gently rinsed 2x with LB3 (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.01% NP-40, protease cocktail inhibitor) without disturbing the pellet. Nuclei were resuspended in 1 mL LB3 and transferred to a 1 ml milliTUBE (Covaris). Chromatin was sheared to a range of 300-600 base-pair fragments using a Covaris E220 evolution Focused ultrasonicator with the following parameters: Peak power (140.0), Duty Factor (5.0), Cycles/Burst (200), Duration (600 seconds), Temperature (4°C). Sheared chromatin was quantified by Bradford Assay, and 450 µg of chromatin was brought to 500 µL in LB3 and 500 µL of ChIP Cocktail Mix (40 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.5% NP-40, Protease inhibitor cocktail) was added. Prepared chromatin was pre-cleared by incubation with 20 µL of pre-washed Dynabeads Protein G magnetic beads (Invitrogen, 10004D) for 3 hours at 4°C with rotation. After bead removal, 10% input (100 µL) of pre-cleared chromatin was removed and set aside. Pre-cleared chromatin was immunoprecipitated with 5 µL of H3K27me3 antibody (Cell Signaling 9733) or 5 ul of H3K9me3 (Active Motif 39161) overnight at 4°C with constant rotation. Protein G magnetic beads (35 µL/IP) were blocked in buffer containing PBS, 0.5% BSA, and 20 µg Herring Sperm DNA (Sigma, D7290) with rotation at 4°C overnight. Blocked beads were washed 3X with PBS and 0.5% BSA and 2X with WB1 (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 1% Triton X-100). Immuno-chromatin complexes were incubated with blocked beads for 3 hours with rotation at 4°C. Bead-immuno-chromatin complexes were then washed 3X for 5 min with rotation at 4°C with WB1, 3X with WB2 (50 mM Tris-HCl pH 7.6, 500 mM NaCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 1% Triton X-100), 2X with WB1, and 1X with Low Salt TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 50 mM NaCl). Beads were incubated in 50 µl of Elution Buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA, 150 mM NaCl, 5 mM DTT, 1% SDS) at 65°C for 15 min in 50 µL volume to elute immuno-chromatin complexes. The elution step was repeated, and eluates combined. Eluents and input were incubated overnight at 65°C with constant shaking to reverse crosslinks, followed by incubation at 37°C for 1 hour with DNase-free RNase A, then at 37°C for 2 hours with 10 µL of Proteinase K (20 mg/mL stock). DNA was isolated with a 1.5x ratio of KAPA Pure Beads (KAPA Biosystems KK8000) to DNA volume. Immunoprecipitated fragments and saved inputs were quantified with a Qubit dsDNA High Sensitivity Assay kit (Invitrogen Q32851), and 10 ng of purified DNA for each IP and input sample were used for library preparation with the KAPA Hyper Prep Kit (Kapa Biosystems KR0961). TAZ and Combo treatments required two IPs per biological replicate to attain enough material for library preparation, and this doubling has been accounted for in the parameters for siQ-ChIP (88,89) for these samples. Library preparation including fragment end-repair, A-tail extension, and adapter ligation was conducted per the manufacturer's instructions (KAPA). Adapter-ligated fragments were amplified with 11 cycles following the recommended thermocycler program, and DNA was purified with two rounds of purification using KAPA Pure Beads (KK8000). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Individually indexed libraries were pooled and 75 bp, paired end sequencing was performed on an Illumina NextSeq 500 sequencer using a 150 bp HO sequencing kit (v2) (Illumina Inc., San Diego, CA, USA) or 50 bp, paired end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S2, 100 bp sequencing kit to a minimum read depth of 50M read pairs per IP library and 100M read pairs per Input library.