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| Status |
Public on Nov 01, 2020 |
| Title |
ATAC_Tn5_Control |
| Sample type |
SRA |
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| Source name |
CCE ESC
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| Organism |
Mus musculus |
| Characteristics |
strain: CCE
tissue: embryonic stem cells
genotype: wildtype
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| Treatment protocol |
NA
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| Growth protocol |
WT (CCE) and Eomes-null (CCE, clone 6A6) 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. At D4, EBs were dissociated, stained with Flk-1-APC (Cat# 17-5821-81) and PdgfRa-PECy7 (Cat# 25-1401-82) antibodies and FACS sorted.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tagmentation and indexing of single cell suspensions (6x104 cells) from three independent differentiations of WT (CCE) and Eomes-null (clone 6A6, CCE) day 4 Flk-1hi/PdgfRa- cells was performed as previously described(Buenrostro et al., 2013), using cells from equivalent samples to those used for the RNA-seq experiments described above. To control for sequence bias of the Tn5 transposase, 100ng genomic DNA of WT (CCE) ESCs was also tagmented and indexed.
Libraries were purified with two rounds of Agencourt AMPure XP bead cleanup (Agencourt, 1.5× bead:sample ratio). Library size and concentration were determined using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent). Samples were sequenced using a 75-cycle paired end Nextera kit with custom Nextera index primers taken from Table S1 in Buenrostro et al. 2013(Buenrostro et al., 2013) on the Illumina HiSeq4000 platform.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Paired-end reads were aligned to the mouse genome assembly mm10 using Bowtie2(Langmead and Salzberg, 2012) with the very-sensitive option, sorted and mitochondrial reads discarded using Samtools(Li et al., 2009) and duplicates removed using Picard (https://broadinstitute.github.io/picard/). Bigwig files were generated using deepTools2(Ramirez et al., 2016).
Biological replicates were randomly downsampled to contain the same number of reads for each individual sample and peaks in each sample called using MACS2(Zhang et al., 2008) using the Tn5 control as a control.
MACS2 called peaks with a p-value < 10-3 were used in downstream analyses. Significant changes in ATAC-Seq datasets were identified using the DiffBind package (http://bioconductor.org/packages/release/bioc/html/DiffBind.html) using bam files and an integrative bed file of all identified peaks in each sample with the following filters: FDR < 0.05, fold change > 1.5.
Genome_build: mm10
Supplementary_files_format_and_content: [.bw] bigWig represent genome coverage generated using Deeptools2.
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| Submission date |
Nov 06, 2019 |
| Last update date |
Nov 02, 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) |
| GSE139998 |
The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors [ATAC-Seq] |
| GSE140005 |
The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors |
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| Relations |
| BioSample |
SAMN13221346 |
| SRA |
SRX7106611 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4151329_Tn5_control.bw |
273.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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