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| Status |
Public on May 15, 2024 |
| Title |
O1 AThi |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
cell type: purified hematopoietic stem cell
cell type: hematopoietic stem cell
genotype: Gfplc3/Gfplc3
autophagy status: AThi (GFPlo)
treatment: steady state isolation
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HSCs were sorted into 400 µl staining media, washed with 1 ml PBS and spun down at 500 g for 5 min at 4°C. Liquid was carefully aspirated and pellets were resuspended in 50 µl ATAC-seq lysis buffer (ATAC-RSB [1M Tris-HC1 pH 7.4, 5M NaCl, and 1M MgCl2 in water] with 0.1% Tween 20, 0.01% digitonin and 0.1% NP40) and left on ice for 3 min. Samples were then washed with 1 ml ATAC Wash buffer (ATAC-RSB with 0.1% Tween 20), inverted to mix, spun for 10 min at 500 g at 4°C, and resuspended in 20 µl ATAC transposition buffer (Nextera TD Buffer (Illumina) with 100 nM Nextera Transposase (Illumina), 0.01% Digitonin and 0.01% Tween 20 in PBS). DNA was transposed for 30 min at 37°C, purified using the ZYMO clean and concentrator-5 kit (Zymo), eluted in 22 µl Zymo Elution buffer, then stored at -20°C until library preparation.
Libraries were pre-amplified with a common Ad1 adapter and unique Ad2 adapter sequences, and additionally amplified using qPCR cycle determination with the NEBNext® High-Fidelity 2X PCR Master Mix (NEB). All libraries were prepared on the same day to limit library preparation-associated batch effects. Double sided AMPure XP bead purification kit (Beckman Coulter) was utilized to remove primer-dimers and large fragments > 1000bp. Libraries were QC using Qubit and Agilent Bioanalyzer at the CUMC Herbert Irving Comprehensive Cancer Center Molecular Pathology core.
Samples were sequenced by GENEWIZ using paired end 150bp sequencing on a HiSeq400 instrument to a target depth of 100 million unique reads per sample. Sequencing quality control was performed using FastQC and MultiQC
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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 |
Nextera sequencing adapter contamination was removed using the Trim Galore Cutadapt wrapper in paired end mode
BWA was used to align samples to the GRCm38 ver 104 genome. BAM files were quality controlled by looking at the distribution of reads aligning to chromosomes and the distribution of insert sizes.
Peaks were called using MACS2, removing PCR duplicates. NarrowPeak files generated by MACS2 were quality controlled for distribution of reads mapping to GRCm38 genomic regions using the R CHIPseeker package and Tx.Db.mmusculus.UCSC.mm39.refGene annotation.
For differential ATAC-seq, a GRanges consensus peak object was generated using ChIPQC containing non-redundant open regions present in any sample. 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.
For pathway enrichment analysis, we annotated promoter proximal peaks within ± 1000bp of the transcription start site (TSS) using TxDb.Mmusculus.UCSC.mm39.refGene. For AThi oHSC, ATlo oHSC, and yHSC samples, we used GSEA analysis using the GSEA software and MSigDB Hallmark pathways.
Assembly: GRCm38 ver 104 genome
Supplementary files format and content: BAM files for each sample
Supplementary files format and content: BigWig files for each sample
Supplementary files format and content: Macs2 narrowpeak files for each sample
Supplementary files format and content: csv files containing differentially accessible peaks for each pairwise comparison
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| Submission date |
Apr 21, 2023 |
| Last update date |
May 15, 2024 |
| Contact name |
Paul Vincent Dellorusso |
| E-mail(s) |
paul.dellorusso@gmail.com
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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 (2) |
| GSE229137 |
Autophagy engaging and non-engaging hematopoietic stem cells |
| GSE230275 |
yHSC AThi oHSC ATlo oHSC ATAC-Seq - Differences in Chromatin Accessibility in Autophagy engaging vs non-autophagy engaging old hematopoeitic stem cells (HSC) comapred to young HSC. |
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| Relations |
| BioSample |
SAMN34284839 |
| SRA |
SRX20044838 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7211178_O1-ATHI.last.bw |
93.3 Mb |
(ftp)(http) |
BW |
| GSM7211178_O1-ATHI.last_peaks.narrowPeak.gz |
1.8 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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