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| Status |
Public on Jul 01, 2024 |
| Title |
HBV-specific CD8+ T cells, sample 1, mRNA-derived library |
| Sample type |
SRA |
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| Source name |
Peripheral blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Peripheral blood
cell type: HBV-specific CD8+ T cells
library type: mRNA
antibodies/tags: none
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
HBV-specific CD8+ T cells were enriched by magnetic bead-based enrichment, and surface staining was performed. Cells were stained with barcoded antibodies together with the surface staining solution. The antibody concentrations used were 1 µg per million cells, as recommended by the manufacturer (BioLegend). After staining, cells were washed three times in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 x g 5 min at 4 °C) and supernatant exchange. After the final wash, the cells were resuspended in PBS, filtered through 40 µm cell strainers and processed for sorting. Live HBV-specific CD8+ T cells were sorted in 1,5mL microcentrifuge tubes (Eppendorf, Germany) containing 20µl PBS using BD Aria Fusion (BD, Germany). The 1,5mL microcentrifuge tubes were coated with 5% BSA over night and rinsed twice with 1mL PBS before usage. Naive, CD45RA+CCR7+, T cells were excluded.
Single-cell RNA sequencing was performed using 10x Genomics with feature barcoding technology to multiplex cells from different donors so that they could be loaded on one well to reduce costs and minimize technical variability. Hashtag oligos were obtained as purified and already oligo-conjugated in TotalSeq-C (5’ chemistry) format from BioLegend. Sorted cells were processed through the 10x Genomics V(D)J single-cell workflow according to the manufacturer’s instructions. Libraries were pooled to desired quantities to obtain appropriate sequencing depths as recommended by 10x Genomics and sequenced on a NovaSeq 6000 flow cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
poly A RNA
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| Data processing |
Pre-processing of sequencing results to generate count matrices (gene expression and HTO barcode counts) was performed using the 10x genomics Cell Ranger pipeline.
Further processing was done with Seurat (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis based on gene expression).
Assembly: Alignment was performed using prebuilt Cell Ranger human reference GRCh38 (GENCODE v32/Ensembl 98).
Supplementary files format and content: RNA counts (HBV1_RNA_counts.csv.gz and HBV2_RNA_counts.csv.gz) and HTO counts (HBV1_HTO_counts.csv.gz and HBV2_HTO_counts.csv.gz) are in zipped csv format.
Supplementary files format and content: Datasets were merged and analyzed by Seurat and the analyzed object is in rds format (seurat_object_combined.rds).
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| Submission date |
Feb 26, 2024 |
| Last update date |
Jul 01, 2024 |
| Contact name |
Sagar - |
| E-mail(s) |
sagar@uniklinik-freiburg.de
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| Organization name |
University Medical Center Freiburg
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| Department |
Department of Internal Medicine II
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| Lab |
Sagar
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| Street address |
Hugstetter Straße 55
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| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE259231 |
Attenuated effector T cells are linked to control of chronic HBV infection |
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| Relations |
| BioSample |
SAMN40136721 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8111774_HBV1_RNA_counts.csv.gz |
8.9 Mb |
(ftp)(http) |
CSV |
| Raw data not provided for this record |
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