{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["charles girardot"],"instrument_platform":["Illumina MiSeq","NextSeq 2000"],"study_type":["methylation profiling by high throughput sequencing"],"organism":["Mus musculus"],"species":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16659"],"description":["To quantify the contribution of specific transcription factor binding versus chromatin context in chromatin opening, we inserted libraries containing hundreds of CREs into a landing pad within a neutral chromatin environment, devoid of activating or repressive chromatin modifications, in mESCs using Recombination-Mediated Cassette Exchange (RMCE). Chromatin accessibility of the inserted fragments was then profiled by targeted SMF using PCR primers that anneal to a synthetic flanking sequence, allowing unambiguous differentiation of the ectopic site from its endogenous counterpart. To investigate whether H3K27Ac contributes to chromatin accessibility at enhancers, we globally reduced H3K27Ac levels by chemically inhibiting the histone acetyltransferase p300 with the small molecule A-485 (24 hours treatment, final concentration: 3 μM). This was performed in the same mESCs carrying the CREs at the landing pad. The dataset generated includes amplicon-based SMF data from the ectopic site in DMSO- and A-485-treated conditions in mouse cells (i.e., TC-1 knock-out of the three DNA methyl transferases (DNMT TKO) mESCs). Two biological replicates were generated for each condition. In summary, cells were collected for SMF, which marks accessible cytosines via recombinant methyltransferases, followed by bisulfite sequencing to infer protein-DNA interactions and chromatin accessibility at single-molecule resolution. The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit and sequenced on an Illumina platform, using either a MiSeq 250 bp paired-end run, a MiSeq i100 250 bp paired-end run, or a NextSeq 2000 P1 300 bp paired-end run. Reads were pre-processed with TrimGalore and a custom R script was used to trim the plasmid backbone from the reads. After, pre-processed reads were aligned using QuasR. Further analyses were conducted using custom scripts available at https://github.com/Krebslabrep/TF-chromatin.git."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Single Molecule Footprinting (SMF) was performed as previously described [Kleinendorst et al., 2021a; Sönmezer et al., 2021]. In short, cultured cells were harvested using trypsin and washed twice with 1x PBS. Cells were counted and 250,000 cells were used per reaction. Cell pellets were resuspended in ice-cold lysis buffer, incubated on ice for 10 min and spun at 1,000x g at 4°C for 5 min. Nuclei were resuspended in 1x M.GpC buffer (NEB, #M0227L). For the GpC methyltransferase treatment, freshly made GpC methyltransferase mix (1x M.GpC buffer, 300 mM sucrose, 64 μM SAM (NEB, #B9003S)) and M.CviPI (NEB, #M0227L) were added and incubated at 37°C for 7.5 min. Prewarmed stop solution and proteinase K were added and incubated overnight at 55°C. The next day, DNA was extracted using phenol-chloroform and treated with RNase A at 37°C for 30 min.","Sequencing - Paired-end sequencing on Illumina NextSeq 2000 (flowcell AAFMCVWM5), run mode: 316-6-316","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.","Library Construction - For making the library for targeted SMF at the ectopic site, the extracted DNA was bisulfite converted using the Qiagen Epitect bisulfite kit as described in [Kleinendorst et al., 2021a]. Bisulfite converted DNA was amplified using KAPA HiFi Uracil+ (Roche) ([95 °C, 4 min] × 1; [98 °C, 20 s; 60 °C, 15 s; 72 °C, 20 s] × 35, [75 °C, 5 min] × 1; 4 °C, hold) and purified using AMPure XP beads (0,75x, Beckman Coulter). The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit.","Sequencing - Paired-end sequencing on Illumina MiSeq i100 (flowcell BWR97814-1409), run mode: 311-6-311","Growth Protocol - We use previously described TC-1 ES cells that have a landing pad for targeted insertion using Recombination Mediated Cassette Exchange (RMCE). For targeted insertion of the DNA libraries into the mouse genome RMCE was used, as previously described [Krebs et al., 2014]. Shortly, TC-1 DNMT TKO ES cells were selected under hygromycin (250 μg/ml, Roche, Switzerland) for 14 days. After, 50M cells were nucleofected (Amaxa 4D-Nucleofector Core Unit, Lonza) with 300 µg DNA libraries containing plasmid and 180 µg pIC-CRE plasmid. After 2 days, cells were selected with 3 µM ganciclovir (Selleckchem, Catalog No. S1878) for 10 days."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Valentina Baderna","charles girardot","Arnaud Krebs"],"additional_accession":[]},"is_claimable":false,"name":"Amplicon based single molecule footprinting in TC-1 DNMT TKO mESCs after RMCE and after 24h p300 inhibition","description":"To quantify the contribution of specific transcription factor binding versus chromatin context in chromatin opening, we inserted libraries containing hundreds of CREs into a landing pad within a neutral chromatin environment, devoid of activating or repressive chromatin modifications, in mESCs using Recombination-Mediated Cassette Exchange (RMCE). Chromatin accessibility of the inserted fragments was then profiled by targeted SMF using PCR primers that anneal to a synthetic flanking sequence, allowing unambiguous differentiation of the ectopic site from its endogenous counterpart. To investigate whether H3K27Ac contributes to chromatin accessibility at enhancers, we globally reduced H3K27Ac levels by chemically inhibiting the histone acetyltransferase p300 with the small molecule A-485 (24 hours treatment, final concentration: 3 μM). This was performed in the same mESCs carrying the CREs at the landing pad. The dataset generated includes amplicon-based SMF data from the ectopic site in DMSO- and A-485-treated conditions in mouse cells (i.e., TC-1 knock-out of the three DNA methyl transferases (DNMT TKO) mESCs). Two biological replicates were generated for each condition. In summary, cells were collected for SMF, which marks accessible cytosines via recombinant methyltransferases, followed by bisulfite sequencing to infer protein-DNA interactions and chromatin accessibility at single-molecule resolution. The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit and sequenced on an Illumina platform, using either a MiSeq 250 bp paired-end run, a MiSeq i100 250 bp paired-end run, or a NextSeq 2000 P1 300 bp paired-end run. Reads were pre-processed with TrimGalore and a custom R script was used to trim the plasmid backbone from the reads. After, pre-processed reads were aligned using QuasR. Further analyses were conducted using custom scripts available at https://github.com/Krebslabrep/TF-chromatin.git.","dates":{"release":"2026-05-21T00:00:00Z","modification":"2026-05-21T01:00:39.342Z","creation":"2026-02-13T14:13:11.337Z"},"accession":"E-MTAB-16659","cross_references":{"ENA":["ERP189115"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0002761","EFO_0005518","EFO_0004184"]}}