|
| Status |
Public on Dec 31, 2023 |
| Title |
R2-only_FL_PAP_2 |
| Sample type |
SRA |
| |
|
| Source name |
primary foetal liver erythroid cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: R2-only mice derived from C57Bl8 mouse strain
tissue: Foetal liver cells (E12.5)
|
| Growth protocol |
To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul per 10^7 cells, following the manufacturer’s instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for ChIP-Seq as in Hay D et al, Nature Genetics, 2016.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
To isolate foetal liver erythroid cells, HetXHet crosses were established. Pregnant mothers were sacrificed at embryonic day 12.5. Foetuses (WT and mutant) were isolated and the foetal liver extracted. Erythroid cells were disaggregated and collected for further processing.
For RNA-seq libraries, 1-2 μg of total RNA was depleted of rRNA using the ribo-Zero rRNA Removal Kit (Illumina) according to the manufacturer’s instructions. To enrich for mRNA, poly(A)+ RNA was isolated, strand-specific cDNA was synthesised, and the resulting libraries prepared for Illumina sequencing using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs: E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs: E7760) following the manufacturer’s instructions. Poly(A)+ and Poly(A)- RNA-seq libraries were sequenced on the Illumina Nextseq platform using a 75-cycle paired-end kit (NextSeq 500/550 High Output Kit v2.5: 20024906).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
R2-only_vs_WT_featurecounts_PAP.txt
|
| Data processing |
Mapping: STAR
Duplicate removal: samtools rmdup
Assembly: mm9
Supplementary files format and content: BAM
|
| |
|
| Submission date |
Nov 30, 2022 |
| Last update date |
Dec 31, 2023 |
| Contact name |
Mira Kassouf |
| E-mail(s) |
mira.kassouf@imm.ox.ac.uk
|
| Organization name |
Weatherall Institute of Molecular Medicine
|
| Lab |
Doug Higgs
|
| Street address |
John Radcliffe Hospital
|
| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE219062 |
Super-enhancers require enhancers and facilitators to fully activate gene expression [RNA-seq] |
| GSE220463 |
Super-enhancers require enhancers and facilitators to fully activate gene expression |
|
| Relations |
| BioSample |
SAMN31943950 |
| SRA |
SRX18429161 |
