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Sample GSM3100973

Query DataSets for GSM3100973

Status

Public on Dec 01, 2018

Title

H3K27ac ChIP-seq, BM-hMSC-TERT4 osteoblast diff 7d rep1

Sample type

SRA

Source name

BM-hMSC-TERT4 osteoblast diff 7d

Organism

Homo sapiens

Characteristics

cell line: BM-hMSC-TERT4
differentiation status: osteoblast
timepoint: 7d
chip antibody: H3K27ac (ab4729, Abcam)

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

Chromatin was prepared from 150 mm culture dishes first by crosslinking in 1% formaldehyde in PBS (10 minutes, RT). Cross-linking was stopped by adding glycine to a final concentration of 0.125 M (10 minutes, RT). The cells were washed twice in ice-cold PBS, and harvested in ice-cold lysis buffer (0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris pH=8) and sonicated at high setting in a Bioruptor-Twin (Diagenode) at a volume of 1.5 ml in 15-mL tubes for 40 cycles of 30 seconds on and 30 seconds off. The chromatin IPs were performed as described in (Nielsen R, et al. 2014. Methods in Enzymology 537: 261-279). Chromatin used for ChIP-seq of MED1 was also cross-linked in 2 mM disuccinimidyl glutarate (DSG) for 20 minutes at RT prior to cross-linking with formaldehyde.
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

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 1500

Description

H3K27ac_7dOb_BM_rep1
H3K27ac_7dOb_BM.bedGraph.gz
H3K27ac_7dOb_BM.bigWig

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