H3K27ac ChIPseq of the ecDNA-positive colon cancer cell line COLO 320DM with and without hydroxyurea treatment
H3K27ac ChIPseq of the ecDNA-positive colon cancer cell line COLO 320DM with and without hydroxyurea treatment
COLO320DM cells were washed 2x with prewarmed 1X PBS. Cells were fixed by addition of 10mL ice-cold 1% FA in 10% FCS-RPMI-1640 and incubated for 10 min on a shaker. The fixation reaction was quenched by addition of 550uL ice-cold 2.5M Glycine in PBS. Cells were washed 1x with ice-cold 1X PBS before scraping down in 10 mL ice-cold 1X PBS. Fixed cells were centrifuged at 500 x g for 5 min at 4°C and resuspended in ice-cold 1X PBS for a total of 2 washes. Cells were pelleted and snap frozen in liquid nitrogen and stored at -80 C for no more than 1 week. Cell pellets were resuspended in 5 mL ice-cold lysis buffer 1 (140 mM NaCl; 50 mM HEPES824 KOH pH 7.5; 1 mM EDTA; 10% Glycerol (v/v); 0.5% IGEPAL CA-630 (v/v); 0.25% Triton 825 X-100 (v/v); 1x Protease inhibitor (Roche, 04693132001)) and incubated on a roller at 4°C for 10 min. Lysed cells were centrifuged at 1357 x g for 5 min, resuspended in 4 mL lysis buffer 2 (20 mM NaCl; 10 mM Tris-HCl pH 8.0; 2 mM EDTA; 0.5M EGTA; 1x Protease inhibitor) and incubated on a roller for 10 min at room temperature. The lysate was centrifuged at 1357 x g for 5 min at 4°C and resuspended in 900 μL sonication buffer (100 mM NaCl; 10 mM Tris HCl pH 8; 1 mM EDTA; 0.5 EGTA; 0.1% Na-deoxycholat (w/v); 0.5% N-Laroylsarcosine (w/v); 1x Protease inhibitor). Lysed chromatin was sheared to a fragment size of 200-500 base pairs (bp) on a Covaris S220 (PIP 140, DF 5, CPB 200) for 12 min. Sheared chromatin was clarified by addition of 1/10 volume 10% Triton X-100 (v/v) and centrifugation at maximum speed at 4°C. Protein-DNA-complexes were immunoprecipitated by overnight incubation with the respective primary antibody on a rotator at 4°C. 10-15 μg total chromatin was used per sample. 30 μL Protein G beads (Invitrogen, 10003D) were washed 3x in 0.25% BSA in PBS (w/v) and added to each sample. Samples were incubated overnight on a rotator at 4°C. Antibody-bead - complexes were washed in RIPA buffer (50 mM HEPES-KOH pH 7.5; 1mM EDTA; 1% IGEPAL CA-630 (v/v); 0.7% Na-deoxycholat (w/v); 500 mM LiCl; 1x Protease inhibitor) for a total of 7 times followed by 1 wash in TE buffer (10 mM Tris-HCl pH 8.0; 1mM EDTA; 50 mM NaCl; 1x Protease inhibitor). Beads were eluted in 200 μL Elution Buffer (50 mM Tris-HCl pH 8.0; 10 mM EDTA, 1% SDS (v/v)). 20 μL 5M NaCl and 5 μL Proteinase K (NEB, P8107S) were added to each sample and DNA was de-crosslinked at 65°C overnight, followed by RNA digestion with 2 uL RNase A (Invitrogen, R1253) at 37°C for 30 minutes. DNA was purified using the QIAquick PCR Purification Kit (Qiagen, 28104) according to the manufacturer's instructions.  ChIP DNA libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645S) and barcoded for Illumina sequencing using the NEBNext Multiplex Oligos for Illumina kit (NEB, E7780S.