|
| Status |
Public on Dec 09, 2022 |
| Title |
All-HE2 |
| Sample type |
SRA |
| |
|
| Source name |
Hemogenic Endothelium 1 (HE2) cells derived from HM-1 ES cell line
|
| Organism |
Mus musculus |
| Characteristics |
cell line: HM-1 ES
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 |
total RNA |
| Extraction protocol |
7x10^6 cells from the all-cytokine and 2.8x10^6 from the -VEGF blast cultures were were sorted for scRNA-Seq library preparation. Cells were stained with CD41-PECY (1:100), KIT-APC (1:100) and TIE2-PE (1:200) and sorted into HE1 (KIT+, TIE2+, CD41-) and HE2 (KIT+, TUE2+, CD41+) populations. Cells were resuspended in 80μl at a concentration of 100-1200 cells/μL for evaluation of viability prior to loading 10,000 cells per sample on a Chromium Single Cell Instrument (10x Genomics)
Library generation for scRNA-Seq was performed by the genomics Birmingham sequencing facility using the Chromium Single Cell 3' library and gel bead kit v3 (10x Genomics). Libraried were paired-end sequenced on an Illumina Novaseq 6000 using the cycle parameters recommended by 10x Genomics
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Read 1 (R1) - Cell barcode and unique molecular identifier (UMI). Read 2 (R2) Biological read
|
| Data processing |
Fastq files from scRNA-Seq experiments were aligned to the mouse genome (mm10) using CellRanger count v6.0.1
Gene expression was measured as UMI counts per cell using gene models from Ensembl (release 102) as the reference transcriptome
Assembly: mm10
Supplementary files format and content: HDF5 feature barcode matrix files generated using CellRanger Count.
|
| |
|
| 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 |
GPL24247 |
| Series (2) |
| GSE198774 |
A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification (Single cell RNA-Seq) |
| GSE198775 |
A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification |
|
| Relations |
| BioSample |
SAMN26725075 |
| SRA |
SRX14474590 |
