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Sample GSM3405915 Query DataSets for GSM3405915
Status Public on Dec 01, 2018
Title ATAC-seq, Primary stromal cells from MUS undifferentiated donor b
Sample type SRA
Source name Primary stromal cells from MUS undifferentiated donor b
Organism Homo sapiens
Characteristics cell line: Primary stromal cells from MUS
differentiation status: undifferentiated
timepoint: n/a
Treatment protocol Immortalized cells and non-mesenchymal cells: 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.
Human primary stromal cells: Primary cells were expanded prior to the induction of differentiation with DMEM/F-12 supplemented with 10 % FCS, 1 % P/S, 15 mM HEPES and 1 nM FGF-1. Osteogenic induction was similar as for the immortalized cells while the adipogenic induction cocktail contained DMEM/F12 supplemented with 1 % P/S, 15 mM HEPES, 500 µM isobutylmethylxanthine, 100 nM dexamethasone, 10 µg/mL insulin and transferrin, 2 nM T3 and 200 mM rosiglitazone. Both induction cocktails were replaced every third day, while dexamethasone and isobutylmethylxanthine were withdrawn at day three and rosiglitazone at day 6 of adipogenic differentiation
Growth protocol Telomerase-immortalized human mesenchymal stromal cells of bone marrow (BM-hMSC-TERT4) or adipose tissue origin (AT-hMSC-TERT) as well as non-mesenchymal cells were grown under standard cell culture conditions in α-MEM supplemented with 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (P/S). Primary stromal cells were gron in DMEM/F12 supplemented with 15 mM HEPES, 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (P/S)
Extracted molecule genomic DNA
Extraction protocol ATAC-seq: Cells were scrabed off from 6-well dishes in ice cold PBS, centrifuged and permeabilised. Nuclei were rinsed through a cell strainer (10 micro m) and counted. 5.000 nuclei were exposed to 1 micro l transposase as described originally (Buenrostro J D, et. al 2015. Current Protocols in Molecular Biology 109: 1-9).
ATAC-seq libraries were constructed according to the manufacturer's instructions (Illumina).
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina HiSeq 1500
Description ATAC_HumanPrimaryStromalCells.bed
Data processing Quality of all libraries were assessed using FastQC ( /projects/fastqc/).
ATAC-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.
Sequencing reads from donors of a tissue of origin were pooled to indentify regions enriched of open chromatin using HOMER. ATAC- regions for Human Primary Stromal Cells were merged using Homer with distance option -d given.
ATAC-seq signal was quantified using HOMER (Heinz S, et al. 2010. Molecular Cell 38: 576-589) with the options -noadj. Normalization of sequencing reads was performed using DEseq2 (Love M I, et al. 2014. Genome Biology 15: 550) taking Donors into the design matrix.
Genome_build: hg19
Supplementary_files_format_and_content: Bed files contain genomic regions identified from ATAC-seq experiments in the immortalized cells and in human primary stromal cells. Peak column in bed files indicate with 1 if the peak was found in the specific samples, 0 indicates absence. Txt file contains non-adjusted gene expression values for eauch replicate and averaged normalized counts and standard deviation for each time point as well as FDR and Log2FC with respect to undifferentiated state for human primary stromal cells. Txt file contains non adjusted quantification of ATAC-seq libraries from human primary stromal cells for two individual donors, averaged normalized counts as well as standard deviations.
Submission date Sep 28, 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 SAMN10142170

Supplementary data files not provided
Processed data are available on Series record
Raw data not provided for this record

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