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| Status |
Public on Sep 22, 2021 |
| Title |
WT EBd7 CD71+ H3K4me1 2 |
| Sample type |
SRA |
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| Source name |
E14TG2a.IV mES derived Erythroid cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: E14TG2a.IV mES derived Erythroid cells
strain: 129/Ola
chip antibody: H3K4me1: Abcam ab8895 at 0.3ug/ml
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| Extracted molecule |
genomic DNA |
| Extraction 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.
Cells were fixed with 1% Formaldehyde, lysed, sonicated and immunoprecipitated with the antibody of interest (CTCF: Merck Millipore 07-729 at 5ug/ml, H3K27ac: Abcam ab4729 at 0.5ug/ml, H3K4me1: Abcam ab8895 at 0.3ug/ml, H3K4me3: Abcam ab8580 at 2.5ug/ml). Libraries were prepared using the NEB Ultra II library preparation kit according to manufacturer’s instructions. Samples were sequenced using 40 bp paired end reads on the NextSeq platform
The strategy was to characterise the chromatin state of 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Chromatin from purified CD71+ erythroid cells derived from differentiated WT mES cells, ChIPed for H3K4me1, sample 2
Genome-wide library of ChIPed chromatin
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| Data processing |
Trim_galore (to remove sequencing adaptors)
FLASH (to reconstruct paired end reads into single reads where possible)
Alignment to the genome (Bowtie) maintaining strict read order
Removal of PCR duplicates;
Combining data from multiple replicates (custom scripts)
Removal of ploidy regions
Genome_build: mm9
Supplementary_files_format_and_content: BigWig
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| Submission date |
Sep 20, 2021 |
| Last update date |
Sep 23, 2021 |
| Contact name |
Mira Tony Kassouf |
| E-mail(s) |
mira.kassouf@imm.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Weatherall Institute of Molecular Medicine
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| Lab |
Doug Higgs
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| Street address |
John Radcliffe Hospital, Headly way
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| City |
Oxford |
| State/province |
England |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE184434 |
Scalable Production of Mouse Erythroid Cells using an in vitro Embryoid Body differentiation System [ChIP-Seq] |
| GSE184435 |
Scalable Production of Mouse Erythroid Cells using an in vitro Embryoid Body differentiation System |
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| Relations |
| BioSample |
SAMN21516534 |
| SRA |
SRX12260415 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5588967_EB_CD71_H3K4me1_ChIP_2.bw |
206.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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