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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 01, 2020 |
| Title |
ChIP_WT_IP |
| Sample type |
SRA |
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| Source name |
Day 4 EBs
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/Sv//Ev
tissue: mixed mesoderm
genotype: wildtype
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| Treatment protocol |
NA
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| Growth protocol |
WT (CCE) and EomesV5/V5 (clones A and B) ESC lines were maintained in feeder free culture conditions in serum + LIF as previously described(Costello et al., 2011). 48-72 hrs prior to induction of hematopoietic differentiation cells were washed with PBS and cultured in serum free ES media containing 50% Neurobasal Media (Gibco, Cat # 21103049), 50% DMEM/F12 (Gibco, Cat # 11320033), supplemented with 0.5X of both N2 (Gibco, Cat#17502048) and B27 (Gibco, Cat# 17504044), 1%Pen/Strep, 1% glutamine, 0.05% BSA (Gibco, Cat# 15260037), 1 uM PD0325091, 3 uM CHIR99021 and 1000 U/ml LIF. At D0 cells were dissociated using Trypsin-LE (Gibco) and seeded at a density of 1×10^5 cells ml in serum-free differentiation (SF-D) media(Nostro et al., 2008) and cultured on an orbital shaker at 70 rpm for ~18 hrs in the absence of growth factors to form EBs. At D2, EBs were split 1:3 in SF-D media containing recombinant human (rh) VEGF (5 ng ml-1; R&D Systems), rhBMP4 (10 ng ml-1; R&D Systems) and Activin A (5 ng ml-1; R&D Systems) for 48 hr.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Day 4 EomesV5/V5 (CCE) and WT (CCE) D4 EBs were dissociated using Trypsin-LE (Gibco) and ~3-4x107 cells were cross-linked for 10 min at RT with 1% (v/v) formaldehyde, followed by quenching with 125mM glycine. Nuclei were recovered and lysed to obtain chromatin, which was then sonicated to 200–500 bp, pre-cleared with protein G Dynabeads (Thermofisher Scientific) and ~175 ug of chromatin was immunoprecipitated with 10μg of anti-V5 (Abcam cat# ab9116, lot# GR: 322448-4) bound to protein G Dynabeads overnight on a rotator at 4ºC. Dynabeads were washed and immune complexes were eluted in IP elution buffer (1% SDS, 0.1M NaHCO3), reverse crosslinked overnight at 65ºC, RNaseA treated for 1.5hrs at 42ºC, proteinase K treated for 2hrs at 45ºC and DNA was recovered using a Zymo column kit.
DNA was multiplexed and paired end sequencing was performed on a single lane of an Illumina HiSeq4000 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
CHIP_WT.bw
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| Data processing |
Paired-end reads were aligned to the mouse genome assembly mm10 using Bowtie2(Langmead and Salzberg, 2012) with default parameters.
PCR duplicates were removed using Samtools 1.9(Li et al., 2009).
Peaks were called against input DNA using MACS 2.1.2(Zhang et al., 2008) and were considered significant if they had a fold enrichment > 2 and FDR < 0.05. Bedtools 2.27.1(Quinlan and Hall, 2010) was used to intersect the peak files from both EomesV5/V5 clones amd subtract peaks called in the WT control and genomic intervals located in mm10 blacklist regions (https://www.encodeproject.org/files/ENCFF547MET/@@download/ENCFF547MET.bed.gz), generating a peak file containing 338 genomic intervals (Supplementary Table 6) used for downstream analyses.
Bigwig files were generated using Deeptools2 (Ramirez et al., 2016)
Genome_build: mm10
Supplementary_files_format_and_content: [.bw] bigWig represent genome coverage (subtract input control) generated using Deeptools2.
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| Submission date |
Nov 06, 2019 |
| Last update date |
Nov 01, 2020 |
| Contact name |
Luke Thomas Gilbert Harland |
| E-mail(s) |
luke.harland@path.ox.ac.uk
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| Organization name |
Oxford University
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| Department |
Sir William Dunn School of Pathology
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| Lab |
Elizabeth Robertson
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3RE |
| Country |
United Kingdom |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE140000 |
The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors [ChIP-Seq] |
| GSE140005 |
The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors |
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| Relations |
| BioSample |
SAMN13221332 |
| SRA |
SRX7106600 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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