{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["charles girardot"],"instrument_platform":["Element AVITI"],"study_type":["ChIP-seq"],"organism":["Mus musculus"],"species":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16466"],"description":["To confirm the loss of transcription factor (TF) occupancy at SNP-containing motifs, we performed ChIP-seq for three TFs in the Bl6xSpret hybrid mESC line. This dataset includes cross-link ChIP-seq data for CTCF, KLF4, and SOX2. Two biological replicates were generated for each TF.   The protocol was adapted from Trovato et al. (2024), with minor modifications. Chromatin was sheared using a Bioruptor Pico (Diagenode) and incubated overnight at 4 °C with rotation with antibodies against KLF4 (AF3158, R&D Systems), CTCF (07-729, Merck Millipore), or SOX2 (AF2018, R&D Systems). Bead–immunocomplexes were reverse cross-linked after RNA and protein digestion, and DNA was purified using 1.4x SPRI-select beads. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs) and sequenced on an AVITI platform, using Cloudbreak Low 2x150 bp runs (250 M clusters/run) for H3K27Ac and Cloudbreak High 2x75 bp runs (1000 M clusters/run).  AVITI and Illumina adapters were trimmed using TrimGalore! v0.6.7 (Krueger et al., 2023). Trimmed reads were competitively aligned to the Bl6 and Spret genomes and filtered to discard reads with allelic mapping bias using a version of WASP extended to include indels (van de Geijn et al., 2015; Sigalova et al., 2025). Finally, duplicate reads were identified and removed using the Picard tool MarkDuplicates v2.15.0. (https://github.com/Krebslabrep/TF-chromatin.git)."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Sequencing libraries were sequenced on Aviti Cloudbreak Low 2x150 (250 M clusters/run) for H3K27Ac and Aviti Cloudbreak High 2x75 (1000 M clusters/run) for the TFs.","Sample Collection - Mouse ES cells (129 WT, DNMT TKO, TET TKO and F1 hybrid cells (129/CAST)) were cultured on 0.2% gelatin-coated plates in ES medium (DMEM, supplemented with 15% Fetal Bovine Serum (FBS), LIF, 2-Mercaptoethanol, 2 mM L-Glutamine and 1x non-essential amino acids) at 37°C and 5% CO2. Medium was changed daily and cells were split every second day.","Nucleic Acid Extraction - The protocol was adapted from (Trovato et al., 2024), with minor modifications. 10 million mESCs (i.e. DNMT TKO cells for H3K27Ac and Bl6xSpret F1 hybrid cells for TFs) were harvested and cross-linked in 3 ml pre-tempered (25°C) ES medium containing 1% formaldehyde and supplemented with sodium butyrate (5mM final concentration) for 10 min at room temperature with rotation. The cross-linking reaction was quenched by adding glycine (125mM final concentration) and incubated for 10 min at room temperature. Cells were washed twice with ice-cold PBS containing 10% FBS and centrifuged at 200g for 5 min at 4°C. At this point, cell pellets can be snap frozen and stored at –80 °C for several months. Pellets were resuspended in 300 µl Sonication Buffer (50 mM Tris-HCl pH 8.0; 0.5% SDS) and chromatin was sheared using the Bioruptor Pico (Diagenode) for 15 cycles (30” ON/30” OFF). Sonicated lysates were diluted 1:6 in lysis buffer (10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 1% Triton X-100; 1 mM EDTA; 0.5 mM EGTA; 0.1% sodium deoxycholate; and 0.5% N-lauroylsarcosine). After centrifugation at full speed for 10 min at 4 °C, the soluble fraction was collected. A 2.5% portion of the supernatant was kept as input, and the fragmentation pattern (~150–500 bp) was analyzed by agarose gel electrophoresis. At this point, sonicated lysates may be aliquoted and stored at –80 °C with a final concentration of 10% glycerol. For each immunoprecipitation (IP), 30 µl of Protein G Dynabeads (Invitrogen) were washed twice with 1 ml of PBS-T (PBS + 0.01% Tween-20) and incubated with 3 µg antibody against H3K27ac (ab4729, Abcam), 5 µg antibody against KLF4 (AF3158, R&D Systems), CTCF (07-729, Merck Millipore) or Sox2 (AF2018, R&D Systems) for 1 h at room temperature with rotation. Coated beads were washed once in cold PBS-T and twice in lysis buffer, then resuspended in 30 µl of lysis buffer per IP and added to chromatin (15 µg for H3K27Ac; 10 µg for TFs). At this point, for the H3K27Ac ChIP, 1 µg of exogenous chromatin (from Drosophila Schneider 2 (S2) cells, prepared with the same protocol and stored at –80 °C with a final concentration of 10% glycerol) was added to each reaction as spike-in. After overnight incubation at 4 °C with rotation, bead-immunocomplexes were washed twice (5 min per wash), using the following buffers: RIPA, RIPA supplemented with 360 mM NaCl, and LiCl buffer (10 mM Tris-HCl, pH 8.0; 250 mM LiCl; 0.5% NP-40; 0.5% sodium deoxycholate; 1 mM EDTA). The complexes were then briefly rinsed with TE buffer and eluted in ChIP SDS elution buffer (10 mM Tris-HCl, pH 8.0; 300 mM NaCl; 5 mM EDTA; 0.5% SDS). RNA and protein digestion were performed by adding 2 µl of RNase A (10 mg/ml stock) and incubating for 30 min at 37 °C, followed by the addition of 1.5 µl of Proteinase K (20 mg/ml stock) and incubation at 55 °C for 1 h. Cross-links were reversed by overnight incubation at 65 °C. DNA was then purified using 1.4X SPRI-select beads.","Library Construction - Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs)"],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Colm Niall Doyle","Valentina Baderna","charles girardot","Arnaud Krebs","Guido Barzaghi"],"additional_accession":[]},"is_claimable":false,"name":"ChIP-seq in Bl6xSpretus F1 hybrid mESCs","description":"To confirm the loss of transcription factor (TF) occupancy at SNP-containing motifs, we performed ChIP-seq for three TFs in the Bl6xSpret hybrid mESC line. This dataset includes cross-link ChIP-seq data for CTCF, KLF4, and SOX2. Two biological replicates were generated for each TF.   The protocol was adapted from Trovato et al. (2024), with minor modifications. Chromatin was sheared using a Bioruptor Pico (Diagenode) and incubated overnight at 4 °C with rotation with antibodies against KLF4 (AF3158, R&D Systems), CTCF (07-729, Merck Millipore), or SOX2 (AF2018, R&D Systems). Bead–immunocomplexes were reverse cross-linked after RNA and protein digestion, and DNA was purified using 1.4x SPRI-select beads. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs) and sequenced on an AVITI platform, using Cloudbreak Low 2x150 bp runs (250 M clusters/run) for H3K27Ac and Cloudbreak High 2x75 bp runs (1000 M clusters/run).  AVITI and Illumina adapters were trimmed using TrimGalore! v0.6.7 (Krueger et al., 2023). Trimmed reads were competitively aligned to the Bl6 and Spret genomes and filtered to discard reads with allelic mapping bias using a version of WASP extended to include indels (van de Geijn et al., 2015; Sigalova et al., 2025). Finally, duplicate reads were identified and removed using the Picard tool MarkDuplicates v2.15.0. (https://github.com/Krebslabrep/TF-chromatin.git).","dates":{"release":"2026-05-08T00:00:00Z","modification":"2026-05-08T01:01:43.307Z","creation":"2025-12-22T16:48:57.026Z"},"accession":"E-MTAB-16466","cross_references":{"ENA":["ERP186919"],"EFO":["EFO_0002944","EFO_0004170","EFO_0002692","EFO_0005518","EFO_0004184"]}}