{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Jan-Inge Bjune"],"organism":["Mus musculus"],"software":["R studio"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13524"],"description":["A map of global Irx3 binding sites during early stages of adipogenic differentiation.  C57BL/gNJ (B6N) mice were fed a SDS maintenance chow diet, and at the age of 6-10 weeks, gWAT and iWAT was isolated and primary preadipocytes were isolated. For each depot, cells from 6-12 mice were pooled. Isolated cells were cultured until confluence in 2D and induced to differentiate 2 days post confluence by a standard adipogenic cocktail.  One day before (day -1) and one day after (day 1) induction of differentiation, cells were fixated by formaldehyde, treated with protease inhibitors, snap-frozen and shipped to Active Motif for ChIP-seq analysis.   Reads were aligned to mouse genome (mm10) using Bowtie2 (version 2.3.4.3), with duplicates and low quality mapping reads (<30) removed via samtools. Peak files were annotated using the R package ChIPseeker (version 1.22.0). Log2 ChIP-over-input tracks for each alignment were generated using deepTools bamCompare (version 3.1.2), and blacklisted regions from Encode were excluded. Bam files were submitted to Macs2 (version 2.1.1) for peak calling. A q-value of 1E-4 and 10X enrichment was used as threshold."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Nucleic acid library construction was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52.","Sequencing - Nucleic acid sequencing was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52.","Sample Collection - Mouse inguinal and gonadal white adipose tissues where excised from 6-10 weeks old C57BL/6NJ (B6N) mice fed ad libitum with SDS maintenance chow (RM3, 3.6 kcal/g).  Primary preadipocytes were isolated from the WAT depots by by collagenase digestion and centrifugation to separate floating adipocytes from the SVF pellet. The SVF was resuspended in DMEM GlutaMax culture medium supplemented with 10% FBS and 1% PEST. The cell suspension was filtered through a 40 um mesh and pooled cells from 6-12 mice were seeded on a 10 cm dish at a density of 100.000 cells/mL.  Cells were grown to confluence, and induced to differentiate 2 days post confluency by addition of 0.5 mM IBMX, 1 uM Dex, 5 ug/mL human Insulin to the growth media.  On days -1 and 1 of differentiation, cells were fixated by addition of 1/10 volume of freshly prepared formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1mM EDTA pH 8.0 and 50 mM HEPES pH 7.9) to the culture media. After 15 minutes, the fixation reaction was stopped by addition of 1/20 volume of 2.5 M glycine. The cells were thereafter kept on ice and washed twice with 0.5% Igepal in PBS. In the second wash, 1mM PMSF was added. Pellets were snap-frozen in liquid nitrogen and stored at -80oC until shipment on dry ice to Active Motif for ChIP-sequencing","Nucleic Acid Extraction - Nucleic acid extraction was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Reads were aligned to mouse genome (mm10) using Bowtie2 (Version 2.3.4.3). Resulting filtered alignment files were first filtered to include only reads that were the primary alignment, with any duplicates and supplementary alignments being removed via samtools. Reads with a mapping quality below 30 were also removed. Filtered read sets ranged from 25 to 31.5 million reads.  Peak files were then annotated using the R package ChIPseeker (Version 1.22.0) in order to annotate peak location in relation to known genomic features, as well as to generate maps of ChIP peak binding in reference to transcription start sites where peaks were within 3000 base pairs of a TSS. Log2 ChIP-over-input tracks for each alignment file were generated using deepTools bamCompare (Version 3.1.2), filtering out any reads from the Encode blacklist [Encode, mm10, 2014], and then uploaded to a UCSC track hub. Bam files were also submitted to Macs2 (Version 2.1.1) for peak calling, providing input data for each day as control. A q-value filter of 1e-4 was used to filter low-confidence peaks from the resulting Macs2 peak files. A total peak set of all bam files in the first replicate, as well as a merged signal graph track, were created for visualization."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NA","Illumina","Illumina HiSeq 2000"],"study_type":["ChIP-seq"],"species":["Mus musculus"],"pubmed_authors":["Jan-Inge Bjune"],"additional_accession":[]},"is_claimable":false,"name":"ChIP-seq of Irx3 in primary cells from mouse gWAT and iWAT on days -1 and 1 of adipogenic differentiation","description":"A map of global Irx3 binding sites during early stages of adipogenic differentiation.  C57BL/gNJ (B6N) mice were fed a SDS maintenance chow diet, and at the age of 6-10 weeks, gWAT and iWAT was isolated and primary preadipocytes were isolated. For each depot, cells from 6-12 mice were pooled. Isolated cells were cultured until confluence in 2D and induced to differentiate 2 days post confluence by a standard adipogenic cocktail.  One day before (day -1) and one day after (day 1) induction of differentiation, cells were fixated by formaldehyde, treated with protease inhibitors, snap-frozen and shipped to Active Motif for ChIP-seq analysis.   Reads were aligned to mouse genome (mm10) using Bowtie2 (version 2.3.4.3), with duplicates and low quality mapping reads (<30) removed via samtools. Peak files were annotated using the R package ChIPseeker (version 1.22.0). Log2 ChIP-over-input tracks for each alignment were generated using deepTools bamCompare (version 3.1.2), and blacklisted regions from Encode were excluded. Bam files were submitted to Macs2 (version 2.1.1) for peak calling. A q-value of 1E-4 and 10X enrichment was used as threshold.","dates":{"release":"2025-05-16T00:00:00Z","modification":"2024-11-26T12:02:11.101Z","creation":"2023-11-03T21:30:20.711Z"},"accession":"E-MTAB-13524","cross_references":{"ENA":["ERP153190"],"Biostudies":["E-MTAB-13525","E-MTAB-13520"],"EFO":["EFO_0002944","EFO_0004170","EFO_0002692","EFO_0005518","EFO_0003816","EFO_0004184"]}}