biostudies-arrayexpressOnur DenizMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15119The intent of the study was to profile the methylome and hydroxymethylome in intestinal stem cells with young mitochondria or old old mitochdondria, to ascertain the potential role of mitochondrial age specific influence on epigenetic regulation of ISCs.biostudies-arrayexpressNucleic Acid Extraction - DNA was isolated using the Quick-DNA Microprep Plus Kit(D4074) according to manufacturers instructionsSample Collection - Intestinal stem cells enriched for Old or Young mitochondria were FACS sorted. Cells form 3-4 mice of both genders(designated with M or F in sample metadata) were pooled for one biological replicate, to a total of four biological replicates.Library Construction - 5-mC and 5-hmC modifications were determined using oxidative bisulfite sequencing(oxBS-Seq) with the NuGEN Ultralow Methyl-Seq with TrueMethyl®oxBs(M01512;Tecan). 300 ngs of genomic DNA were sonicated using a Covaris S220 sonicator(Covaris) to a mean size of 250-300bp. For quantitative assessment of oxidation the DNA was spiked with 1% of control DNA-duplexes, containing C, 5-mC and 5-hmC bases at known positions. The sonicated DNA from was split into two aliquots of 150ngs, one was oxidized, and bisulfite converted, the other was mock oxidized before bisulfite conversion. oxBS libraries required 10 PCR amplification cycles, BS libraries required 8 cycles. Library quality was assessed on an Agilent TapeStation D5000(Agilent Technologies, Santa Clara, CA).Sequencing - libraries were normalized to 10nM, pooled, clustered on a pair-end-read flow cell and sequenced for 150 cycles on an Illumina NovaSeq600, obtaining about 300 million clusters per library.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw data was quality controlled and aligned to mouse genome build GRCm38.p6(mm10).Data Transformation - Methylation calls were generated using the Epigenomics Core in-house bisulfite sequencing analysis pipeline. Methylation sites with a minimum read coverage of 10 were selected for the following analysis. 5-hydroxymethylation was obtained by subtracting the OxBS from the BS frequencies. Methylation data for CpGs was processed using the methylKit(1.33.1) in R. DMCs had a minimum methylation difference of 25% and a q-value cutoff of 0.01.Unbiased GO-term enrichment analysis was performed based on statistically signifanct DMC and DhMCs assocaiting with genes using pantherdb GO-molecular function complete. DMRs were obtained using Metilene(0.2-8) similarly to enzymatic 5hmC, but with no estimation of missing data and with -M300 and -M500 for 5mC and 5hmC, respectively. DMRs were annotated using ChIPseeker with gene models from TxDb.Mmusculus.UCSC.mm10.knownGene. Paneth cell vs ISCs mRNA fold changed of the DMRs associating with genes in ISC mito-O was obtained from RNA-sequencing data (Pentinmikko et al. 2019) Arrayexpress;E-MTAB-7916.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Bisulfite-seqMus musculusOld mitochondria regulate niche renewal via alpha-ketoglutarate metabolism in stem cellsOnur DenizSimon AnderssonSimon Andersson, Hien Bui, Arto Viitanen, Daniel Borshagovski, Ella Salminen, Sami Kilpinen, Angelika Gebhart, Emilia Kuuluvainen, Swetha Gopalakrishnan, Nina Peltokangas, Martyn James, Kaia Achim, Eija Jokitalo, Petri Auvinen, Ville Hietakangas, Pekka KatajistofalseWhole-genome oxidative bisulfite sequencing and data processing of mouse intestinal stem cells with old and young mitochondriaThe intent of the study was to profile the methylome and hydroxymethylome in intestinal stem cells with young mitochondria or old old mitochdondria, to ascertain the potential role of mitochondrial age specific influence on epigenetic regulation of ISCs.2025-05-07T00:00:00Z2025-05-07T21:36:54.503Z2025-05-06T20:12:08.481ZE-MTAB-15119ERP172394EFO_0002944EFO_0004170EFO_0003753EFO_0004917EFO_0005518EFO_0003816EFO_0004184