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| Status |
Public on Jun 16, 2023 |
| Title |
ATAC-seq_D2_unsort_FABV_3 |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: mouse embryonic stem cells
day: Day 2
treatment: Pretreatment
sort: Unsorted
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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 cells were 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 10 cm 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 2 mM L-glutamine, 1 mM ascorbic acid (MilliporeSigma), and 10 ng/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 |
Total RNA was extracted using NucleoSpin RNAII kit (Clonetech). ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) was performed as previously described (Buenrostro et al. 2015).
For RNA-seq, library preparation was performed by the University of Chicago Genomics Facility using the Illumina TruSeq RNA Sample prep kit v2 (Part #RS-122-2001). Library fragments were approximately 275bps in length and were quantitated using the Agilent Bio-analyzer 2100 and pooled in equimolar amounts. Single-ended, 51bp sequencing was performed on the Illumina HiSeq2500 in Rapid Run Mode by the University of Chicago Facility. For ATAC-seq, libraries were amplified and normalized with the Illumina Nextera DNA Library prep kit (FC-121–1031) according to the manufacturer's protocols. Libraries were quantitated using the Agilent Bioanalyzer, pooled in equimolar amounts, and sequenced with 50-bp single-end reads on the Illumina HiSeq following the manufacturer's protocols through the Genomics Core Facility at the University of Chicago.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Transcripts were aligned to the indexed reference for mm10 with default settings using TopHat2 v2.1.1 (Kim et al. 2013).
Reads were filtered using bamtools using the following settings: -isDuplicate false -mapQuality “>10” (Barnett et al. 2011).
Transcript reads were counted post-alignment using StringTie (Pertea et al. 2015).
Differential expression testing was performed using edgeR v3.16.5 (Robinson and Smyth 2007; Robinson and Smyth 2008; Robinson et al. 2010; McCarthy et al. 2012; Zhou et al. 2014) and limma v3.30.13 (Ritchie et al. 2015) packages in R v3.3.2.
Low level genes were removed within each condition using median log2-transformed counts per gene per million mapped reads (cpm) of 1 and a union generated from those lists.
Differential expression testing was performed using a general linear model (GLM) framework. For comparing FAB and FAB+V, a covariate for replicate was included to correct for batch effect.
Sequencing reads were aligned to the mm10 genome using Bowtie2 v2.3.0 (Langmead and Salzberg 2012) and SAMtools v0.1.19 (Li et al. 2009; Li 2011).
Peak calling was performed using MACS2 callpeak (Zhang et al. 2008; Feng et al. 2012) using the settings --nomodel --shift -100 --extsize 200 -q 0.1 after pooling biological replicates.
A fold-enrichment track was generated using MACS2 using the bdgcmp function (-m FE) for visualization on the genome browser.
Following removal of ENCODE blacklist sites (Amemiya et al. 2019; ENCODE Project Consortium 2012), a union set of sites was generated by identifying summits that overlapped within 200bp and arbitrarily selecting the summit of highest positional value. Summits were then extended by 200bp in both directions to create the set of union sites.
Fold-enrichment scores were assigned to each site using the multiBigwigSummary function from deepTools (Ramírez et al. 2016).
Genome_build: mm10
Supplementary_files_format_and_content: RNAseq_Compilation.txt contains all outputs related to the RNA-seq datasets, including TPM values for every biological replicate and differential expression testing. TSS_TAD.bed contains the TSS and TAD assignments. UnifiedATAC_Dataset.txt contains a list of the union sites, positional information, and fold-enrichment scores. edgeR_Results_FABVoverFAB.txt is the differential accessibility testing comparing the FAB+V and FAB conditions. All files "_summits_noBlacklist.bed" contain the summit calls from MACS2 following removal of ENCODE blacklist sites. All "_FE.bw" are bigWig files containing the MACS2 fold-enrichment scores.
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| Submission date |
Aug 30, 2019 |
| Last update date |
Jun 17, 2023 |
| Contact name |
Jeffrey D. Steimle |
| E-mail(s) |
[email protected], [email protected], [email protected]
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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 |
GPL17021 |
| Series (1) |
| GSE136692 |
ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment |
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| Relations |
| BioSample |
SAMN12662573 |
| SRA |
SRX6784682 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4055368_06_D2_unsort_FABV_3_FE.bw |
186.8 Mb |
(ftp)(http) |
BW |
| GSM4055368_06_D2_unsort_FABV_3_summits_noBlacklist.bed.gz |
688.5 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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