|
| Status |
Public on Sep 28, 2023 |
| Title |
human CD4+ T cells_CD73-_biol rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
Peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
donor: D3
tissue: Peripheral blood
cell type: blood-derived CD4+ T cells
sorted cell type: CD73negative
|
| Treatment protocol |
Freshly isolated CD4+ T cells from healthy blood donors were sorted based on their CD73 expression. To that purpose, cells were stained with APC anti-Human CD73 (1:50, Biolegend) diluted in PBS and incubated for 20 min at RT according to the manufacturer's protocol. Cells were washed and kept on ice. Fluorescence-activated cell sorting was performed using the MA900 Multi-Application Cell Sorter (Sony Biotechnologies). 3x10^6 CD73- and CD73+ CD4+ T cells were collected respectively, and stored as dry cell pellets at -80 °C until further processing.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated from three donors by Ficoll-Hypaque density gradient centrifugation (Corning) at 2000 rpm (~ 850 x g) at RT for 30 min, without brake. PBMCs were immediately processed to isolate CD4+ T cells by negative selection using the EasySep Human CD4+ T Cell Isolation Cocktail (StemCell Technologies) according to manufacturer’s protocol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA preparation was conducted at Genewiz (USA).
library preparation was conducted at Genewiz (USA).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
3-D3-CD73neg
raw_counts.csv
normalized_counts.csv
rlog_transformed_counts.csv
|
| Data processing |
Paired-end sequencing was performed using the Illumina NovaSeq 6000 instrument to obtain a minimum of 20 million read pairs per sample with a read length of 2x150 bp.
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36
The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2.
The hit counts were summarized and reported using the gene_id feature in the annotation file. Only unique reads that fell within exon regions were counted.
Using DESeq2, a comparison of gene expression between samples was performed adjusting for the donor effect as confounding variable. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 (Benjamini-Hochberg method) and absolute log2 fold change > 1 were called as differentially expressed genes for each comparison.
Assembly: Homo sapiens GRCh38
Supplementary files format and content: raw counts for each sample
Supplementary files format and content: normalized counts for each sample
Supplementary files format and content: rlog transformed counts for each sample
|
| |
|
| Submission date |
Sep 27, 2023 |
| Last update date |
Sep 28, 2023 |
| Contact name |
Hannah Sabeth Sperber |
| E-mail(s) |
Hannahsperber.7@gmail.com
|
| Organization name |
Essen University Hospital
|
| Department |
Institute for transaltional HIV Research
|
| Lab |
Schwarzer Lab
|
| Street address |
Virchowstr. 171
|
| City |
Essen |
| State/province |
NRW |
| ZIP/Postal code |
45147 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE244190 |
The Hypoxia-regulated Ectonucleotidase CD73 is a Host Determinant of HIV Latency [RNA-seq] |
| GSE244193 |
The Hypoxia-regulated Ectonucleotidase CD73 is a Host Determinant of HIV Latency |
|
| Relations |
| BioSample |
SAMN37567845 |
| SRA |
SRX21908283 |
