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Sample GSM3565074 Query DataSets for GSM3565074
Status Public on Feb 26, 2019
Title ATACseq_serum_free_ctrl_rep2
Sample type SRA
 
Source name differentiating mES cells
Organism Mus musculus
Characteristics tissue: differentiating mES cells
differentiation protocol: serum-free
time: day4 of differentiation
purification: unsorted
doxycycline-treatment: not treated
Growth protocol Mouse ES cell differentiation was achieved by diluting 40000 cells/ml in EB differentiation medium and incubation in an orbital shaker at 80 RPM. EB differentiation medium: IMDM supplemented with 15% FBS , 1% Penicillin/Streptomycin, 2mM Glutamax, 50 μg/ml Ascorbic acid, 4.5 mM Monothioglycerol. For Serum-free differentiation, FBS was replaced with an equivalent amount of Knock-outTM Serum Replacement (Invitrogen). Trangene induction was achieved by adding doxycycline (Sigma-Aldrich) to day 3 EBs cultures (final concentration 1µg/ml), and then maintained throughout the differentiation protocol by replacing the media (including dox) every two days. At day 5, EBs were disgregated and single cells were incubated for 20 minutes with PDGFRα-PE and FLK1-APC conjugated antibodies. PDGFRα+FLK1- cells were sorted using FACSAriaII (BD biosciences) and replated on gelatin coated dishes using serum- or serum-free EB differentiation media supplemented with 1µg/ml doxycycline and 10ng/ml mouse basic-FGF.
Extracted molecule genomic DNA
Extraction protocol ATAC-seq: 50,000 freshly sorted cells from differentiated ES cultures, iPax3 NIH3T3 fibroblasts and single embryos were washed with 200 µl of cold PBS then resuspended in 100 µl of cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630), spun at 500 g for 10 minutes at 4°C and resuspended in 50 µl of the transposition reaction mix. Transposition occurred at 37°C for 30 minutes, after which transposed DNA was purified using a Qiagen MinElute Kit and eluted in 10µl Elution Buffer. Final libraries were generated by primer extension of the transposed DNA followed by PCR amplification using Illumina-compatible adapter-barcodes.
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description processed data file:
Ctrl_KSR_B_ATAC_peaks.bed
serum_free_ctrl_02
Data processing ATAC-seq analysis. Reads were aligned to the mouse genome (mm10) using Bowtie2 with the following parameters: -q -I 38 -X 2000 --local --dovetail --no-mixed --no-discordant. Aligned reads were filtered for PCR duplicates using Samtools and then processes with MACS 1.4 with the following parameters: --nomodel --nolambda --keep-dup all --call-subpeaks -p 1e-4 -w –S. Bigwig files for visualization on IGV were generated by converting the wig files obtained from MACS using the Kent tool Wig-to-BigWig. ATAC-seq reads overlapping to Pax3 peaks were retrieved using the R Bioconductor package and then used to generate density maps with Seqminer. To identify differentially accessible peaks, we used the BEDTools intersect function: the common regions (-f 0.5 -F 0.5 -e options) for the dox-induced samples were intersected (-v -f 0.2 options) with the combined list of control peaks (from non-induced cells) and the resulting list was then compared (-f 0.5 -F 0.5 –e options) with the peak list detected in PDGFRα+FLK1- cells from E9.5 embryos.
Genome_build: mm10
Supplementary_files_format_and_content: ATAC-seq: processed files contain the list of peaks identified by MACS in each replicate
 
Submission date Jan 16, 2019
Last update date Feb 26, 2019
Contact name Alessandro Magli
E-mail(s) alemagli@gmail.com
Organization name University of Minnesota
Department Medicine
Street address 2231 6th St SE
City Minneapolis
State/province MN
ZIP/Postal code 55455
Country USA
 
Platform ID GPL17021
Series (1)
GSE125203 Chromatin accessibility landscape upon induction of Msgn1, Pax3 and Myf5 in mesodermal cells and identification of conserved Pax3 binding sites and target genes during skeletal myogenesis
Relations
BioSample SAMN10756557
SRA SRX5255361

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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