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Sample GSM3100944

Query DataSets for GSM3100944

Status

Public on Dec 01, 2018

Title

DNase-seq, BM-hMSC-TERT4 adipocyte diff 4h rep2

Sample type

SRA

Source name

BM-hMSC-TERT4 osteAdlast diff 4h

Organism

Homo sapiens

Characteristics

cell line: BM-hMSC-TERT4
differentiation status: adipocyte
timepoint: 4h
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_4hAd_BM_rep2
DNase1_4hAd_BM.bedGraph
DNase1_4hAd_BM.bigWig
DNase1_IDR_peaks.bed

Data processing

Quality of all libraries were assessed using FastQC (http://www.bioinformatics.babraham.ac.uk /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

E-mail(s)

s.mandrup@bmb.sdu.dk

Phone

+45 6550 2340

Organization name

University of Southern Denmark