|
| Status |
Public on Dec 09, 2022 |
| Title |
HE2-Serum-R2 |
| Sample type |
SRA |
| |
|
| Source name |
Hemogenic Endothelium 2 (HE2)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: HM-1 ES dervied Hemogenic Endothelium 2 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 |
SAMN26724589 |
| SRA |
SRX14474829 |
