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Sample GSM5956762 Query DataSets for GSM5956762
Status Public on Dec 09, 2022
Title ES-Serum_R2
Sample type SRA
 
Source name Embryonic Stem Cell (ES)
Organism Mus musculus
Characteristics cell type: HM-1 ES cells
mouse strain: 129/ola
Growth protocol Standard ES cell differentiation used serum and the purification of differentiating cells was conducted essentially as described in Obier et al. (2016) Serum Free I.V.D Culture Embryoid bodies (EBs) were generated from HM-1 mouse embryonic stem cells by plating at 5.0x105 cells/ml in serum free (SF) media into petri-grade dishes. BMP4 was added to a concentration of 5ng/ml. Cultures were left to incubate at 37°C and 5% CO2 for 60 hours before bFGF and Activin A were added at a concentration of 5ng/ml each. The cells were incubated for 16 hours at 37°C 5% CO2 and then sorted for FLK1+ cells as described in Obier et al. (2016). FLK1+ cells were plated in serum free media on 0.1% gelatine coated plates or for larger cultures flasks at 2.25x104 cells per cm. BMP4, Activin-A and bFGF were added to a concentration of 5ng/ml for 16 hours. Media was then removed, the blast culture was washed with PBS and fresh SF media was added containing BMP4 (5ng/ml), VEGF (5ng/ml), TPO (5ng/ml), SCF (100ng/ml), IL6 (10ng/ml) and IL3 (1ng/ml). For cytokine withdrawal experiments, one of BMP4, VEGF, IL6 or IL3 was not added at this stage. Blast cultures were left to incubate at 37°C and 5% CO2 for 72 hours before cells were harvested for cell sorting and FACS analysis. Inhibition of trypsin was achieved using Trypsin Inhibitor (Thermofisher) following the manufacturer’s instructions.
Extracted molecule genomic DNA
Extraction protocol Extraction was performed as part of the ATAC-Seq library preparation protcol
ATAC-Seq was performed as described in Buenrostro et al (2015), briefly 5000-50,000 HB, HE1, HE2 and HP cells were sorted by FACS and transposed in 1x tagment DNA buffer Tn5 transposase and 0.01% Digitonin for 30 minutes incubated at 37°C with agitation. DNA was purified using a MinElute Reaction Cleanup Kit and DNA was amplified by PCR using Nextera primers.
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model NextSeq 550
 
Data processing Single-end reads from ATAC-seq experiments were processed with Trimmomatic (version 0.39) and aligned to the mm10 mouse genome using Bowtie2 2.4.4 using the options -very-sensitive-local.
Open chromatin regions (peaks) were identified using MACS2 2.2.7.1 using the options -B -trackline -nomodel.
The peak sets were then filtered against the mm10 blacklist.
Assembly: mm10
Supplementary files format and content: Bed files of ATAC-Seq peak co-ordinates
 
Submission date Mar 16, 2022
Last update date Dec 09, 2022
Contact name Peter Keane
E-mail(s) p.keane@bham.ac.uk
Organization name University of Birmingham
Department Institute for Cancer and Genomic Sciences
Street address Vincent Drive
City Birmingham
ZIP/Postal code B15 2TT
Country United Kingdom
 
Platform ID GPL21626
Series (2)
GSE198772 A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification (ATAC-seq)
GSE198775 A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification
Relations
BioSample SAMN26724592
SRA SRX14474832

Supplementary file Size Download File type/resource
GSM5956762_B486_6-peaks-filtered.bed.gz 1.6 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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