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Sample GSM3100935 Query DataSets for GSM3100935
Status Public on Dec 01, 2018
Title DNase-seq, BM-hMSC-TERT4 osteoblast diff 3d rep1
Sample type SRA
Source name BM-hMSC-TERT4 osteoblast diff 3d
Organism Homo sapiens
Characteristics cell line: BM-hMSC-TERT4
differentiation status: osteoblast
timepoint: 3d
chip antibody: n/a
Treatment protocol Two days post confluency cells were induced to undergo osteoblast or adipocyte differentiation by switched to α-MEM supplemented with 10% FCS, 10 mM dexamethasone, 10 nM vitamin D, 10 mM β-glycerolphosphate, and 50 µg/ml ascorbic acid or to DMEM supplemented with 10% fetal serum, 10ug/mL insulin, 1µM rosiglitazone, 100 mM dexamethasone, and 500 µM isobutylmethylxanthine. Media was replaced on day 2, 4, 7, 9 and 11.
Growth protocol Telomerase-immortalized human mesenchymal stromal cells of bone marrow (BM-hMSC-TERT4) or adipose tissue origin (AT-hMSC-TERT) were grown under standard cell culture conditions in α-MEM supplemented with 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (P/S).
Extracted molecule genomic DNA
Extraction protocol Nuclei were isolated from cells grown on 150 mm dishes using a 0.04% NP-40 buffer (15 mM Tris-HCl pH=8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA pH=8.0, 0.5 mM EGTA pH=8.0, 0.5 mM Spermidine). 10 million freshly isolated nuclei were treated with 40 units/ml DNase I for 3 minutes at 37°C as decribed previously (Siersbaek R, et al.2011. EMBO Journal 30:1459-72). After RNase and Proteinase K treatment, DNA was isolated by phenol-chloroform-extraction and separated by ultra-centrifugation on a sucrose gradient overnight. DNA-fragments between 100 and 500 bp (representing 2-hit cutting events) were isolated, checked for enrichment of open chromatin over background using real- time PCR.
DNase-seq and ChIP-seq libraries were constructed from 10 to 20 ng of genomic DNA according to the manufacturer's instructions (Illumina) as described in (Nielsen R, et al. 2014. Methods in Enzymology 537: 261-279).
Library strategy DNase-Hypersensitivity
Library source genomic
Library selection DNAse
Instrument model Illumina HiSeq 1500
Description DNase1_3dOb_BM_rep1
Data processing Quality of all libraries were assessed using FastQC ( /projects/fastqc/).
ChIP-seq and DNase-seq reads were aligned to the hg19 genome using STAR aligner (Dobin A, et al., Bioinformatics 29:15-21) with the following parameters: --outSJfilterIntronMaxVsReadN 0 --alignIntronMax 1 --alignSJDBoverhangMin 200, with all other parameters set at default. Only one read per position and read length was kept. RNA-seq reads were aligned to hg19 genome using STAR aligner and default settings. Only one read per position and read length was kept.
Regions enriched for open chromatin or MED1 recruitment were identified using the ENCODE IDR (LI Q H, et al. 2011. Annals of Applied Statistics, 5:1752-1779) pipeline and MACS2 peak caller (Zhang Y, et al. 2008. Genome Biology 9:R137). For ChIP-seq samples a pooled Inout control sample was used as control. Reproducible peaks between replicates for the single time points were merged using Homer with distance option -d given.
Gene expression was quantified using HOMER (Heinz S, et al. 2010. Molecular Cell 38: 576-589) with the options -count exons -condenseGenes -noCondensing -noadj. Normalization and differential expression analysis was performed using DEseq2 (Love M I, et al. 2014. Genome Biology 15: 550) taking Replicates and Time Points into the design matrix, excluding genes which were not detected in both cell lines. Log 2 fold changes and FDR values were calculated with respect to undifferentiated MSC.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph and bigWig files were created using HOMER for visualization of enhancer features in genome browser. Bed files contain reproducible genomic regions from IDR analysis of DNase1-seq, MED1 ChIP-seq and their overlap with and without promoters. Txt file contains non-adjusted gene expression values for eauch replicate and averaged normalized counts and standard errors for each time point as well as FDR and Log2FC with respect to undifferentiated MSC for both BM-hMSC-TERT4 and AT-hMSC-TERT4 cells.
Submission date Apr 17, 2018
Last update date Dec 01, 2018
Contact name Susanne Mandrup
Phone +45 6550 2340
Organization name University of Southern Denmark
Department Biochemistry and Molecular Biology
Street address Campusvej 55
City Odense M
ZIP/Postal code 5230
Country Denmark
Platform ID GPL18460
Series (1)
GSE113253 Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis
BioSample SAMN08941771
SRA SRX3946886

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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