ChIPmentation

ChIPmentation was carried out as previously described,78 with minor adaptions. Cells were washed once with PBS and fixed with 11% formaldehyde fixation solution (1M HEPES-KOH, pH 7.5, 5M NaCl, 0.5M EDTA, pH 8.0, 0.5M EGTA, pH 8.0, and 37% formaldehyde and H₂0) in 10 mL PBS for 10 min at room temperature. Glycine was added at a final concentration of 2.5M to stop the reaction. Cells were collected at 500 g for 10 min at 4C (subsequent work was performed on ice and used cool buffers and solutions unless otherwise specified) and washed twice with up to 0.5mL ice-cold PBS supplemented with 1X Halt Protease Inhibitor Cocktail Solution (ThermoFisher 78446). The pellet was lysed in sonication buffer (10mM Tris-HCl pH 8.0, 2mM EDTA pH 8.0, 0.25% SDS, 1X Halt Protease Inhibitor Cocktail Solution) and sonicated with a Covaris LE220 Ultrasonicator for 8 minutes (CH22 parental and engineered chordoma cells) or 4 minutes (UM-Chor1 parental and engineered chordoma cells) in a microtube until the size of fragments was in the range of 200–700 base pairs. Lysates were transferred to new tube and diluted 1:1.5 with equilibration buffer (10mM Tris, 233mM NaCl, 1.66% Triton X-100, 1.66% sodium deoxycholate, 1 mM EDTA, 1X Halt Protease Inhibitor Cocktail Solution). Lysates were centrifuged at full speed for 10 min at 4C and the supernatant containing the sonicated chromatin was transferred to a new tube. 10L of chromatin was removed for the whole cell extract (WCE). The antibody was added to the lysates and incubated on a rotator overnight at 4C. The following antibodies were used: H3K27ac (Cell signaling D5E4), and HA (Cell signaling C29F4) (1μg per one million cells per immunoprecipitation). Magnetic Protein A beads (ThermoFisher 10001D) (10μL per IP) were washed twice and blocked overnight in 1mL of PBS supplemented with 0.1% BSA. Blocked beads were then added to immunoprecipitated lysates and incubated for 2hrs on a rotator at 4C. Beads were washed subsequently with RIPA-LS (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0,140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate and 1X Halt Protease Inhibitor Cocktail Solution) (twice), RIPA-HS (10mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate and 1X Halt Protease Inhibitor Cocktail Solution) (twice) and RIPA-LiCl (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250 mM LiCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% NP40 and 1X Halt Protease Inhibitor Cocktail Solution) (twice). Beads were washed with cold 10mM Tris-HCl pH 8.0, to remove detergent, salts and EDTA. Beads were washed once more with cold 10mM Tris-HCl pH 8.0 and the whole reaction including beads was transferred to a new tube and then placed on a magnet to remove supernatant to decrease background. Beads were then resuspended in 25μL of the tagmentation reaction mix (2X Tagment DNA buffer (Illumina) and water containing 1μL Tagment DNA Enzyme (Illumina)) and incubated at 37C for 7 min in a thermocycler. The beads were washed with RIPA-LS (twice) and with cold TE pH 8.0 (twice) before removing the supernatant. Beads were then incubated with 50mL elution buffer (0.5% SDS, 300mM NaCl, 5mM EDTA and 10mM Tris-HCl pH 8.0) containing 2μL of Proteinase K (ThermoFisher AM2548) for 1h at 55C and 10h at 65C, to revert formaldehyde crosslinking. 10μL of WCE was also de-crosslinked with elution buffer and Proteinase K in a 50μL reaction. Post de-crosslinking, supernatants were transferred to a new tube. Antibody-treated samples were de-crosslinked a second time for 1 hour at 55C in 19μL of ChIP elution buffer and 1μL Proteinase K and then purified with the Zymo DNA Clean and Concentrator (−5) kit (Zymo D4013) and eluted in 27μL of water. WCE DNA was purified with Zymo DNA Clean and Concentrator (−5) kit (Zymo D4013) and eluted in 12.5μL of water before preforming the tagmentation reaction described above and purifying the DNA again (eluting in 27μL of water). Enrichment of the libraries was performed in a 50μL reaction using 0.75mM primers, 25mL NEBNext High-Fidelity 2X PCR MasterMix (M0541) and 20μL of the purified library. Libraries were amplified for N+2 cycles, where N is equal to the rounded-up C_T value determined in a test qPCR reaction with 4μL of the library. Enriched libraries were size selected and purified using SPRI AMPure XP beads (Beckman A63881) at a beads-to-sample ratio of 0.65:1 to recover libraries with a fragment length of 200–400 bp. ChIPmentation libraries were run on an Illumina Nextseq 500 instrument (single-end 75 bp reads).