|
| Status |
Public on Dec 31, 2023 |
| Title |
WT_FL_MED1_1 |
| Sample type |
SRA |
| |
|
| Source name |
primary foetal liver erythroid cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Primary erythroid cells derived from FL
strain: C57BL/6
chip antibody: Bethyl IHC-00149
treatment: Genome-wide ChIPmentation
|
| 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 |
genomic DNA |
| 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.
Cells were fixed with 2mM DSG and 1% Formaldehyde, lysed, sonicated and immunoprecipitated with the antibody of interest. IP'd chromatin was tagmented using Tn5 loaded with illumina sequencing adaptors. Samples were sequenced using 40 bp paired end reads on the NextSeq platform.
The strategy was to characterise the chromatin state of primary mouse-derived or the CD71+ erythroid cells (WT and mutant) derived from mESC-EB in vitro differentitation system by isolating and analysing DNA regions bound by the antibodies of interest, genome-wide.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
genome-wide WT_FL_MED1_1 ChIPmentation in Primary erythroid cells derived from FL derived from C57BL/6 mice.
|
| Data processing |
Trim_galore (to remove sequencing adaptors)
FLASH (to reconstruct paired end reads into single reads where possible)
Alignment to the genome (Bowtie2)
Removal of PCR duplicates;
Combining data from multiple replicates
Removal of ploidy regions
Assembly: mm9
Supplementary files format and content: BAM
|
| |
|
| Submission date |
Dec 07, 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) |
| GSE220456 |
Super-enhancers require enhancers and facilitators to fully activate gene expression [ChIP-seq] |
| GSE220463 |
Super-enhancers require enhancers and facilitators to fully activate gene expression |
|
| Relations |
| BioSample |
SAMN32097398 |
| SRA |
SRX18530091 |
