Histone ChIP-seq for the detection of active elements (H3K4me1, H3K4me3, H3K27ac, input control) in a selection of cancer cell lines
Histone ChIP-seq for the detection of active elements (H3K4me1, H3K4me3, H3K27ac, input control) in a selection of cancer cell lines
10 million cells were crosslinked by incubation with formaldehyde (final concentration 1%; Sigma-Aldrich) for 10 min, followed by quenching with glycine (final concentration 0.125 M; Sigma-Aldrich). Cells were pelleted, washed twice with ice-cold PBS containing fresh cOmplete Protease Inhibitor Cocktail (Roche) and resuspended in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% (v/v) Glycerol, 0.5% (v/v) NP-40, 0.25% (v/v) Triton X-100 and fresh cOmplete Protease Inhibitor Cocktail (Roche) to lyse the plasma membrane. The sample was centrifuged and the supernatant was discarded. The pellet was resuspended in lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and fresh cOmplete Protease Inhibitor Cocktail (Roche). The sample was centrifuged and the supernatant was discarded. Finally, the pellet was resuspended in lysis buffer 3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) Na-deoxycholate, 0.5% (w/v) N-lauroylsarcosine) and fresh cOmplete Protease Inhibitor Cocktail (Roche) to lyse the nuclear membrane and solubilise the chromatin. All cells were maintained in a humidified atmosphere at 37°C with 5% CO2. COV-434 cells were cultured in DMEM, 2 mM Glutamine and 10% FBS. ES-2 cells were cultured in McCoy's 5a and 10% FBS. Kuramochi and SKOV-3 cells were cultured in RPMI 1640 and 10% FBS. OAW28 and OAW42 cells were cultured in DMEM, 2 mM glutamine, 1 mM NaP, 5 mg bovine insulin and 10% FBS. OVISE cells were cultured in DMEM and 10% FBS. TOV-112D cells were cultured in 1:1 MCDB 105 medium plus Medium 199 and 15% FBS. Material was sonicated to yield DNA fragments of 200 – 500 bp using a Bioruptor Pico sonication device (Diagenode) using a cell line-specific cycle number (1 cycle: 30 s ON/30 s OFF, 40 cycles for all cell lines, with the exception of OAW42 and TOV-112D, for which 30 cycles were used). As input control 2% of the chromatin was kept aside and the remainder was used for IP after addition of 6 volumes of RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.1% (w/v) Na-deoxycholate) and fresh cOmplete Protease Inhibitor Cocktail (Roche). ChIP samples were processed according to either the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina Kit or NEBNext Ultra II DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for lllumina (Index Primers Set 1 and 2) with some adjustments explained in the following. For ChIP DNA end repair, 2 µL instead of 1 µL NEBNext End Repair Enzyme Mix was used. Additionally, 2 µL of Klenow Fragment (3´→ 5´exo-) was used instead of 1 µL for dA-tailing of end repaired DNA. Furthermore, annealing/extension and final extension were performed at 72°C instead of 65°C for PCR enrichment of adaptor-ligated DNA. Size selection and primer peak removal was performed using AMPure XP beads at a ratio of 0.9x to ChIP DNA after PCR enrichment instead of before. ChIP DNA was eluted with 26 µL elution buffer.