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| Status |
Public on Jun 16, 2023 |
| Title |
H3K27ac |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
day: Day 4
treatment: FAB+V
sort: P+F+
chip antibody: H3K27ac
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| Growth protocol |
Bry-GFP and ZX1 ES cell lines were maintained in a serum- and feeder-free culture system (Ying et al. 2003; Gadue et al. 2006; Ying et al. 2008; Kattman et al. 2011). Neurobasal medium and DMEM/F12 (50%/50%; Thermo Fisher Scientific) were supplemented with N2 (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), 0.05% BSA (MilliporeSigma), 150mM mono-thioglycerol (MilliporeSigma), 1000unit Mouse LIF (MilliporeSigma), 1μM PD0325901 (Stemgent), 3μM CHIR99021 (Stemgent), L-glutamine (Life Technologies), penicillin-streptomycin (Thermo Fisher Scientific). PD0325901 and CHIR99021 were not added at 2 days prior to start differentiation. For differentiation start, ES cellswere dissociated with trypLE express (Thermo Fisher Scientific) and cultured, at 1.0x105 cells/ml for 48hrs (Gadue et al. 2006; Kattman et al. 2011). In brief, IMDM and Ham’s F12 medium (75%/25%) were supplemented with N2, B27, BSA, L-glutamine, 0.5mM ascorbic acid (MilliporeSigma), 450mM mono-thioglycerol (MilliporeSigma), penicillin-streptomycin. The cells were differentiated in 10cm petri-dishes (Becton Dickenson). The 48hr-old embryoid bodies (EBs) were dissociated with trypLE express and the cells were re-aggregated in StemPro-34 SFM medium (Thermo Fisher Scientific) in the present of L-glutamine, 200μg/ml human transferrin, 500μM ascorbic acid, 450μM mono-thioglycerol, 6ng/ml bFGF, 8ng/ml Activin A and 1ng/ml BMP4 for Bry+, PDGFRα+ and Flk1+ mesoderm induction. 5ng/ml VEGF was treated at D2 for bipotential mesoderm induction. Human Activin A, human BMP4, mouse VEGF, human bFGF were purchased from R&D systems. For hematopoietic lineage and cardiac lineage induction, sorted cells were cultured in StemPro-34 SF medium, supplemented with 2mM L-glutamine, 1mM ascorbic acid (MilliporeSigma), and 10ng/ml bFGF (Kattman et al. 2006; Kattman et al. 2011). 2×105/ml sorted cells were seeded into individual wells of a 48-well flat bottom plate (Becton Dickenson) coated with gelatin after sorting.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells sorted at Day 4 or Day 5 of differentiation and frozen with liquid nitrogen were crosslinked in 0.5% formaldehyde for 5 min at room temperature, and then the reaction was quenched with 125 mM glycine. Cross-linked cells were rinsed with PBST (PBS with 0.1% Tween 20) and then resuspended in LB3-Triton (1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl pH 8, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroyl sarcosine, 1% Triton) supplemented with a protease inhibitor cocktail (Calbiochem 539131). Chromatin was extracted by sonication using Bioruptor (Diagenode). Cell extract was cleared by centrifugation and an aliquot was saved for input DNA sequencing.
Cell extract from 0.5 million cells was incubated with mouse monoclonal anti-H3K27ac antibody (Wako MABI0309, Lot 14007; 2 μL per IP) in a 200 μL reaction for 12 hours or longer at 4oC. Immunocomplex was captured by Protein G-conjugated magnetic dynabeads (ThermoFisher) and washed. Immunoprecipitated DNA was reverse-crosslinked and used to construct high-throughput sequencing libraries using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs). DNA libraries were processed on an Illumina HiSeq machine for paired-end sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Paired-end ChIP-seq reads were aligned to the mouse reference genome mm10 using Bowtie2 with the “--no-mixed --no-discordant -X 1000” parameter set, and then aligned reads with MAPQ >20 were retained.
H3K27ac-enriched peaks were identified using MACS with two biological replicates of ChIP and one biological replicate of the input data, with the default narrow-peak option.
For data visualization, input-normalized per-base fold-enrichment scores were computed using MACS2 with two biological replicates of ChIP and one biological replicate of the input data.
We then computed the sum of the signal coverage of the fold-enrichment data within 50-bp windows across the genome, and the 50-bpwindow data were quantile-normalized across all experimental conditions (Day4 PF–VEGF, Day4 PF, and Day 5 F) using normalize.quantiles function in the preprocessCore package (v1.36.0) in software R.
Assembly: mm10
Supplementary files format and content: Files ending in .bw are bigwig files for data visualization. The per library bigwig files are genome normalized, while the other tracks are the MACS2 input normalized tracks shown in the manuscript.
Supplementary files format and content: Files ending in .narrowPeak are peak calls from MACS2.
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| Submission date |
Mar 17, 2023 |
| Last update date |
Jun 16, 2023 |
| Contact name |
Jeffrey D. Steimle |
| E-mail(s) |
jeffrey.steimle@bcm.edu, jsteimle@uchicago.edu, jsteimle@alumni.nd.edu
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| Phone |
7137985917
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| Organization name |
Baylor College of Medicine
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| Department |
Molecular Physiology and Biophysics
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| Lab |
Room 504E, Mail Stop BCM335
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| Street address |
1 Baylor Plaza
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE136692 |
ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment |
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| Relations |
| BioSample |
SAMN33796489 |
| SRA |
SRX19698695 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7103839_KI948_K27ac_D4PF_ABV_Exp10_mm10_BT2q20xPEn.bw |
48.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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