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| Status |
Public on Jan 30, 2017 |
| Title |
DE_ATAC-seq rep3 |
| Sample type |
SRA |
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| Source name |
Definitive Endoderm
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES_derived Definitive Endoderm
strain: 129/Sv//EV
day of differentiation: Day 5
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| Treatment protocol |
ES cells (ESC) were seeded in suspension at low density (1x104 cells/ml) in the absence of LIF to form embryoid bodies (EBs). ES cells were seeded in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Gibco), 1% Penicillin/Streptomycin (Invitrogen), 0.1mM 2-mercaptoethanol (Sigma), 1% Glutamine (Invitrogen), 1% MEM Non-essential amino acids (Gibco) and 1mM Sodium Pyruvate (Sigma). On day 2 EBs were transferred into N2B27 medium (Cellartis) supplemented with 20ng/ml ActivinA (R&D systems) and 20ng/ml EGF (Peprotech) to induce DE differentiation (Morrison et al. 2008).
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| Growth protocol |
ES cell lines were maintained in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Gibco), 1% Penicillin/Streptomycin (Invitrogen), 0.1mM 2-mercaptoethanol (Sigma), 1% Glutamine (Invitrogen), 1% MEM Non-essential amino acids (Gibco), 1mM Sodium Pyruvate (Sigma), 1000U/ml LIF (ESGRO) on gelatin coated plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013) on single cell suspensions. 60000-80000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to transposition with Tn5 transposase (Nextera, Illumina) for 30 minutes at 37°C. DNA was purified using a MinElute kit (Qiagen).
Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). The libraries were quantified using the universal library quantification kit (KAPA Biosystems).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Genome-wide library of transposed fragments
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| Data processing |
Library strategy: ATAC-Seq
Library Strategy: ATAC-seq
Mapping : bowtie -m 2 --maxIns 350
For un-mapping reads : trimming adaptors trim_galore -- length 10, combining overlapping R1-R2 reads : flash -m 9 -x 0.125
Combining trimmed+flashed and originally mapped reads, mapping again with bowtie -m 2 --maxIns 350
Removing read pairs overlapping Blacklisted regions
Removing duplicates : samtools rmdup
Reconstructing sequenced fragments from R1+R2 read pairs ( bedtools bedpe | cut -f 1,2,6) - for visualisation
Pileup of fragments (bedtools genomecov -counts)
Generating bigwig tracks (ucsctools bedGraphToBigWig)
Genome_build: mm9
Supplementary_files_format_and_content: bigWig : un-normalised coverage of filtered fragments (reconstructed, duplicate-filtered fragments : see above), density of alignments in a moving 300 bp window with an increment of 30 bp
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| Submission date |
Jan 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Damien Downes |
| E-mail(s) |
[email protected]
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| Phone |
01865222374
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| Organization name |
The University of Oxford
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| Department |
MRC Weatherall Institute of Medicine
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| Street address |
John Radcliffe Hospital
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| City |
Headington |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE94249 |
Eomes related gene regulation in Definitive Endoderm (ATAC-seq) |
| GSE94250 |
Eomes related gene regulation in Definitive Endoderm |
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| Relations |
| BioSample |
SAMN06279425 |
| SRA |
SRX2527243 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2471764_DE_C_ATAC.bw |
298.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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