biostudies-arrayexpressJan-Inge BjuneMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13525A map of open chromatin in control and Irx3-KO beige ME3 preadipocytes cells on days 0, 1 and 7 of adipogenic differentiation. Cells were differentiated for indicated number of days, pelleted and snap-frozen in liquid nitrogen. After thawing, cells were treated with DNase to remove exogenous DNA released from cells that died prior to freezing. Remaining cells were lysed in ATAC-seq lysis buffer from the ATAC-seq. Crude nuclear extracts was isolated by centrifugation and resuspended in Tagmentation buffer. Nuclear integrety was verified under microscope, and 50,000 nuclei were transferred for tagmentation. Tagmentation and indexing was performed according to the instructions of the ATAC-seq Kit (Active Motif Catalog No. 53150). Library QC was performed on a Bioanalyzer, and the library was sequenced on a HiSeq4000 Illumina platform using 75 bp paired end sequencing. Reads were filtered to remove low quality mappings, duplicates and mitochondrial mappings. Peaks were called by Macs2 and ENCODE blacklisted regions were excluded. Differentially open chromatin was identified by featurecounts and DESeq2.biostudies-arrayexpressSample Collection - ME3 cells were differentiated for 0, 1 and 7 days, trypsinated, pelleted, snap-frozen in liquid nitrogen and stored at -80C.Library Construction - Tagmentation and indexing was performed according to the instructions of the ATAC-seq Kit (Catalog No. 53150, Active Motif). Briefly, nuclei were incubated in tagmentation buffer for 30 min at 37C with gentle mixing every 5 min. Tagmented DNA was purified by a standard silica spin column method provided by the kit. Indexing/PCR amplification of the library performed using unique combinations of i5 and i7 indexing primers. Indexed DNA was cleaned up using SPRI magnetic beads. Library QC was performed by a D5000 ScreenTape assaySequencing - Sequencing was performed on a HiSeq4000 Illumina platform with 75 bp paired end sequencing.Growth Protocol - Cells were grown in AmnioMAX-C100medium supplementedwith 7.5% FBS, 7.5% C100 (all from Thermo Fisher Scientific,Waltham, MA, USA), 1% penicillin-streptomycin (PEST) (Sigma, St.Louis, MO, USA) and 2 mM L-glutamine (Sigma) at 37 °C and 5% CO2.Adipogenic differentiation was initiated three days post confluency(day 0) by induction medium containing 5 μg/mL Insulin (INS)(Sigma), 1 μMDexamethasone (DEX) (Sigma), 0.5mMisobutylmethylxanthine(IBMX) (Sigma) and 1 μM Rosiglitazone (ROSI) (CaymanChemical, Ann Arbor, MI, USA). From day 2 to day 4 only insulin wasadded to the basal medium and from day 4 to 7 cells were grown inthe basal medium.Nucleic Acid Extraction - The cells were thawed on ice, and treated with DNase to remove DNA released from dead cells before lysis in ice-cold ATAC seq lysis buffer from the ATAC-Seq Kit (Active Motif, Carlsbad, CA, USA) for 2 min. Crude nuclei extracts were spun down at 600xg for 10 min at 4C and nuclei were resuspended in 40 µL 1X Tagmentation buffer. The integrity of the nuclei was verified under microscope, counted, and 50,000 nuclei was transferred pr sample for tagmentation.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Reads were first filtered for mapping quality ≥ 30 (samtools Version 1.7-2) and duplicated reads, as well as reads mapping to the mitochondria, were subsequently removed (picardTools Version 2.8.1.1). Peak calling was performed via Macs2 (Version 2.1.1). Peak sets were first filtered using blacklisted regions from the ENCODE project, then converted into a simple annotation format for submission to the Rsubread featureCounts (Version 1.6.0) package. Using this consensus peak set as an annotation guide, each unmerged bam file was submitted to featureCounts and the subsequent count matrix was input into RStudio for peak annotation and visualization. To detect differential accessibility between control and Irx3-KO samples, individual peaks were first identified using Macs2 as described above, and sequencing reads within each individual peak were then counted using featureCounts and DESeq2.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNAIllumina HiSeq 4000ATAC-seq kit (Catalog No. 53150, Active Motif)ATAC-seqMus musculusJan-Inge BjunefalseATAC-seq of control and Irx3-KO mouse beige ME3 cells on days 0, 1, and 7 of adipogenic differentiationA map of open chromatin in control and Irx3-KO beige ME3 preadipocytes cells on days 0, 1 and 7 of adipogenic differentiation. Cells were differentiated for indicated number of days, pelleted and snap-frozen in liquid nitrogen. After thawing, cells were treated with DNase to remove exogenous DNA released from cells that died prior to freezing. Remaining cells were lysed in ATAC-seq lysis buffer from the ATAC-seq. Crude nuclear extracts was isolated by centrifugation and resuspended in Tagmentation buffer. Nuclear integrety was verified under microscope, and 50,000 nuclei were transferred for tagmentation. Tagmentation and indexing was performed according to the instructions of the ATAC-seq Kit (Active Motif Catalog No. 53150). Library QC was performed on a Bioanalyzer, and the library was sequenced on a HiSeq4000 Illumina platform using 75 bp paired end sequencing. Reads were filtered to remove low quality mappings, duplicates and mitochondrial mappings. Peaks were called by Macs2 and ENCODE blacklisted regions were excluded. Differentially open chromatin was identified by featurecounts and DESeq2.2025-05-16T00:00:00Z2024-11-26T12:01:51.626Z2023-11-03T21:30:56.186ZE-MTAB-13525ERP153192E-MTAB-13524E-MTAB-13520EFO_0002944EFO_0007045EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0004184