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Sample GSM3405919

Query DataSets for GSM3405919

Status

Public on Dec 01, 2018

Title

ATAC-seq, AT-hMSC-TERT undifferentiated rep1

Sample type

SRA

Source name

AT-hMSC-TERT undifferentiated

Organism

Homo sapiens

Characteristics

cell line: AT-hMSC-TERT
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 Both induction cocktails were replaced every third day, while dexamethasone and isobutylmethylxanthine were withdrawn at day three and rosiglitazone at day 6 of adipogenic differentiation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 NovaSeq 6000

Description

ATATC_TERTimmortalizedCells.bed

Data processing

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

E-mail(s)

s.mandrup@bmb.sdu.dk

Phone

+45 6550 2340

Organization name

University of Southern Denmark