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| Status |
Public on May 23, 2021 |
| Title |
O2_PreTx |
| Sample type |
SRA |
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| Source name |
Hematopoietic stem cells
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| Organism |
Mus musculus |
| Characteristics |
age at procedure: 24 months old
transposition day: 1
Sex: Male
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Hematopoietic stem cells were isolated from the bone marrow of mice through a standardized process of bone marrow extraction, creation of a single cell suspension, and flow cytometry based cell sorting. Cells were lysed and transposed following the Corces et al. Nature Methods 2017 Omni-ATAC seq protocol. Transposed DNA was purifed using a Zymo 5 Clean and Concentrator kit and stored at -20 C until library preparation. Libraries were quality controlled using Qubit and Agilent Bioanalyzer.
ATAC-seq libraries were made with Nextera sequencing adapters and NEBnext Master Mix as in Corces et al. Nature Methods 2017. Libraries were purified with double-sided AMPure XP beads.
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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 |
Sequencing quality control was performed using FastQC and MultiQC, which revealed Nextera sequencing adapter contamination that was removed using the Trim Galore Cutadapt wrapper in paired end mode, followed by confirmation of adapter removal with FastQC. For pathway enrichment analysis, we annotated promoter proximal peaks within ± 1000bp of the transcription start site (TSS) using TxDb.Mmusculus.UCSC.mm10.knownGene.
Rsubread was used to align samples to the mm10 genome. Rsamtools was used to sort and index output BAM files. BAM files were quality controlled by looking at the distribution of reads aligning to chromosomes and the distribution of insert sizes.
We next utilized Genrich (Harvard FAS Informatics) to call peaks in ATAC seq mode using a maximum q value threshold of 0.05 for peak calling, removing PCR duplicates and excluding reads that mapped to the mitochondrial genome and the Y chromosome. NarrowPeak files generated by Genrich were quality controlled for distribution of reads mapping to mm10 genomic regions using the R CHIPseeker package and Tx.Db.mmusculus.UCSC.mm10.knowngene annotation.
For differential ATAC-seq, a GRanges consensus peak object was generated using ChIPQC48 containing non-redundant open regions present in any sample, excluding known mm10 ATAC-seq blacklisted regions obtained from Encode (ENCFF547MET). Nucleosome free regions (<100bp fragment length) from sample BAM files were counted using FeatureCounts against the consensus peak annotation.
Sample counts were imported into a dds object using DESeq2, and exploratory data analysis was performed using rlog normalized count data and PCA. For differential peak accessibility calling between groups, we used DESeq2 using the results function and the DESeq2 estimator.
Genome_build: We used ReactomePA to test for Reactome pathway enrichment in peaks with increased relative peak accessibility in a given comparison using a log2 fold change threshold of >= 0.05 and a pvalue cutoff of 0.05.
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| Submission date |
Jun 02, 2020 |
| Last update date |
May 23, 2021 |
| Contact name |
Paul Vincent Dellorusso |
| E-mail(s) |
[email protected]
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| Phone |
5166552491
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| Organization name |
Columbia University Irving Medical Center
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| Department |
Genetics and Development
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| Lab |
Passegue Lab
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| Street address |
701 West 168th Street, 724 Hammer Health Sciences Center
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE151665 |
Young and Old Pre- and Post-Transplantation ATAC Seq |
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| Relations |
| BioSample |
SAMN15084600 |
| SRA |
SRX8455210 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4587843_O2-PreTx.narrowPeak.gz |
780.9 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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