2025-07-31
ENA first public
ERC000011
ENA-CHECKLIST
Erasmus MC Cancer Institute
INSDC center name
public
INSDC status
ERS22952008
SRA accession
E-MTAB-14771:SKOV-3 H3K27ac
Submitter Id
ArrayExpress
broker name
SKOV3
cell_line
disease cell type
cell_type
not collected
collection date
human
common name
Protocols: 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.
description
adult
developmental stage
ovarian carcinoma
disease
EVI1 positive
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
ovary
organism part
Homo sapiens
scientific_name
SKOV-3 H3K27ac
title