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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 04, 2024 |
| Title |
633_E2wd72_rep1_ATACseq |
| Sample type |
SRA |
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| Source name |
63.3 mouse GMP cells
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| Organism |
Mus musculus |
| Characteristics |
treatment: Estrogen withdrawal
time point: 72h
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| Growth protocol |
Cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652), supplemented with 0.5uM beta-estradiol (Sigma, E2758; made from a 10mM stock dissolved in 100% ethanol) and stem cell factor (SCF) from conditioned media generated from a Chinese hamster ovary (CHO) cell line that stably secretes SCF (Sykes et al. Cell 2016; this conditioned media was added at a concentration of 1%). For treatment, cells were washed 2x in PBS and then resuspended in the identical media as above without estrogen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To generate ATAC-seq libraries for 63.3 cells, 50,000 cells were used and libraries were constructed as previously described (Buenrostro et al., 2013; Cheloufi et al., 2015). Briefly, cells were washed in PBS twice, counted and nuclei were isolated from 100,000 cells using 100 μl hypotonic buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) to generate two independent transposition reactions. Nuclei were split in half and treated with 2.5 μl Tn5 Transposase (Illumina) for 30 mins. at 37°C.
DNA from transposed nuclei was then isolated and PCR-amplified using barcoded Nextera primers (illumina). Library QC was carried out using high sensitivity DNA bioanalyzer assay and Qubit measurement and sequenced using paired-end sequencing (PE50) on the Illumina HiSeq 2500 platform.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ATACseq
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| Data processing |
Mouse and human data were trimmed using Cutadapt (v3.4) (with parameters -m 20 -q 20 -a CTGTCTCTTATA -A CTGTCTCTTATA), then aligned to the mm10 and hg38 genomes with Bowtie2 v2.4.2 (with parameters --dovetail -I 10 -X 1000 --very-sensitive).
Reads in the mitochondrial genome and unannotated chromosomes as well as the UCSC blacklist were removed, then duplicates were removed with samtools markdup (v1.1.2), and the insert size distribution was calculated using Picard CollectInsertSizeMetrics (v2.25.2) and validated to contain nucleosomal peaks. BED file was then constructed with each mate pair as a separate read using bedtools bamtobed (v2.3.0), and peaks were called using MACS2 callpeak (v2.2.7.1) (with parameters --nomodel --shift -100 --extsize 200 --keep-dup all --call-summits). Note that the MACS2 command shifts each read by 100 base pairs towards the 5’ end and extends the read length to 200 base pairs, thus treating the 5’ end of the original read (the Tn5 cut site) as the center of a new “fragment” that is then used for peak calling and other downstream analysis.
To normalize tracks, reads overlapping shared peaks were counted for each BAM file using featureCounts (with parameters -M -O) and normalization factors were calculated from these counts using the calcNormFactors function in the edgeR package (v3.32.1) in R. Visualization was performed using the bedGraphToBigWig utility and the igv-jupyter package in Python.
Genome_build: hg38
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files for each sample
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| Submission date |
Apr 19, 2021 |
| Last update date |
May 04, 2024 |
| Contact name |
Brian Do |
| E-mail(s) |
bdo311@gmail.com
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| Organization name |
MIT
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| Department |
Biology
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| Lab |
Vander Heiden
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| Street address |
500 Main St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE172299 |
A myeloid maturation program initiated by nucleotide depletion during S phase [ATAC-Seq] |
| GSE172335 |
Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress |
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| Relations |
| BioSample |
SAMN18794956 |
| SRA |
SRX10632370 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5252118_633_E2wd72_rep1_ATACseq.bw |
300.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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