 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 26, 2019 |
| Title |
ATACseq_serum_ctrl_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
differentiating mES cells
|
| Organism |
Mus musculus |
| Characteristics |
differentiation protocol: serum
time: day4 of differentiation
purification: PDGFRα+FLK1- sorted
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 |
ATAC-seq
|
| 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 |
Feb 26, 2019 |
| Last update date |
Feb 27, 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 |
SAMN11020190 |
| SRA |
SRX5431010 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3629706_Ctrl_EBM_no_A_ATAC_peaks.bed.gz |
583.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
