|
| Status |
Public on May 22, 2024 |
| Title |
bulkATACseq_CD34+_2DG_12h_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Umbilical cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Umbilical cord blood
disease state: Healthy
cell type: CD34+ cells
treatment: 2DG 1mM
|
| Treatment protocol |
Cells were stimulated with cytokines after thawing and were grown in the same medium with or without metabolic inhibitor (2-DG, DON or AOA) until lysis was performed. Cells were collected and lysed after 03h, 12h or 24h of cytokine stimulation.
|
| Growth protocol |
CD34+ cells were purified from human healthy cord blood with a two-step immunomagnetic beads protocol and were then frozen in a cryopreservation medium. After thawing, CD34+ cells were cultured in a 96-well plate in a humidified 5% CO2 incubator at 37°C. Cells were grown in prestimulation medium made of X-Vivo (Lonza) supplemented with penicillin/streptomycin (respectively 100U/mL and 100µg/mL - Gibco, Thermo Scientific), 50 ng/ml h-FLT3, 25 ng/ml h-SCF, 25 ng/ml h-TPO, 10 ng/ml h-IL3 (Miltenyi) final concentration.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Living cells were isolated before transposition by FACS using 7-AAD labeling. Around 5000 cells for each sample were used.
Library was prepared following the Fast ATAC-seq protocol, controled with Bioanalyzer and sequenced with NextSeq or MiSeq system.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
bulk ATAC seq - Fast ATAC protocol
|
| Data processing |
Cleaning and alignment of data using : trimmomatic (v0.32), bowtie2 (v2.4.1), samtools (v1.4), macs2 (v2.1.1), picard (v1.138) and bedtools (v.2.29.2)
Snakemake (v7.25.0) workflow to handle bam files : merge biological donors (samtools merge v1.11), index the files, quality control checking and count reads.
A downsampling step with samtools view function was performed as regard to the smallest number of reads detected in the cohort (80 000 000 reads)
A new peak calling was conducted on downsampled .bam files using the macs2 callpeak tool (v2.2.7.1) with the following parameters: <-f BAMPE -g hs -B –broad –broad-cutoff 0.1>.
R (v4.2.1) & Rsubread (v2.4.0) & GenomicRanges (v1.42.0) & DEseq2 (v1.38.3) & ggplot2 (v3.4.2)
Only peaks with 10 or more reads were kept for further analysis.
Assembly: hg19
Supplementary files format and content: xxx_threshold_10_ann.gr.rds files are genomic ranges object for R scripts. Biological replicates for the same condition and time point were merged and filter on the minimal number of reads per peak was applied.
|
| |
|
| Submission date |
Sep 12, 2023 |
| Last update date |
May 22, 2024 |
| Contact name |
Laëtitia RACINE |
| E-mail(s) |
laetitia.racine@hotmail.fr
|
| Organization name |
EPHE
|
| Lab |
CRSA
|
| Street address |
27 rue de Chaligny
|
| City |
Paris |
| ZIP/Postal code |
75012 |
| Country |
France |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE243003 |
Metabolic adaptation pilots the differentiation of human hematopoietic cells (bulk ATAC-Seq) |
| GSE243006 |
Metabolic adaptation pilots the differentiation of human hematopoietic cells |
|
| Relations |
| BioSample |
SAMN37367062 |
| SRA |
SRX21762732 |
