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| Status |
Public on Apr 27, 2023 |
| Title |
blood.CD4.CD57+.GEX |
| Sample type |
SRA |
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| Source name |
Blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell type: FACS-purified human blood CD4+ CD57+ T cells
genotype: NA (No known genetic defects)
treatment: No treatment
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| Extracted molecule |
total RNA |
| Extraction protocol |
Children with routine tonsillectomy were recruited for tonsils collection, and their blood may be also obtained. After mechanical disruption by surgical blades, tonsillar lymphocytes were collected by gradient centrifugation using Ficoll-Paque Plus tubes (GE Healthcare), and the PBMCs were also collected by gradient centrifugation. CD57+ CD4+ T cells (7AAD- CD19- CD3+ CD4+ CD8- CD57+) and CD57- CD4+ T cells (7AAD- CD19- CD3+ CD4+ CD8- CD57-) from tonsil and blood samples of a child were FACS-purified into 1.5-ml Eppendorf tubes with FACS buffer.
Single cell gene expression and VDJ libraries were prepared by Biomolecular Resource Facility at ANU using Chromium Next GEM Single Cell 5' Kit v2 Dual Index and Single Cell Human TCR Amplification Kit (10x Genomics) according to the manufacturer’s instructions. The library was sequenced in S1 flow cell with 2 x 50 bp paired-end reads by NovaSeq 6000 sequencer (Illumina).
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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 |
10x Genomics-gene expresssion
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| Data processing |
The 10X Cell Ranger package (10X Genomics, v6.0.1) was used to process data and align to human genome (GRCh38-2020-A).
Seurat package (v4.1.0) was utilized for gene expression analyses with a standard processing workflow (Satija et al., 2015). Quality control was performed to filter out the low-quality cells (doublets and dead cells). Cells with the number of detected genes between 200 to 2500 and the percentage of mitochondrion reads less than 5 % were selected for downstream analyses.
Unwanted CD19+ B cells, CD8+ T cells, CD14+ and CD300E+ monocytes (about 1 % in total) were removed from the datasets. Highly variable TRBV and TRAV genes were disregarded for the downstream gene expression and cells cluster analyses. UMI count data were normalized by regularized negative binomial regression (SCTransform).
Samples were then integrated to compare their similarities and differences, anchors were identified with SCTransform.
ScRepertoire package (v1.3.5) was used to analyze TCR clonotypes (Borcherding et al., 2020). VDJ contig outputs from the Cell Ranger pipeline were extracted and combined. Clonal space homeostasis looking at the relative space occupied by clones at specific proportions was performed for tonsillar and blood CD57+ and CD57- CD4+ T cells.
Assembly: GRCh38-2020-A
Supplementary files format and content: Comma-separated values for VDJ sequencing
Supplementary files format and content: Tab-separated values files and matrix files for gene expression
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| Submission date |
Apr 05, 2022 |
| Last update date |
Apr 27, 2023 |
| Contact name |
Yuwei Hao |
| Organization name |
JCSMR of ANU
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| Street address |
Level 6, Building 10, The Canberra Hospital, Yamba Drive,
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| City |
Canberra |
| ZIP/Postal code |
2605 |
| Country |
Australia |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE200257 |
Single-cell RNA-sequencing of blood and tonsillar CD4+ CD57+ and CD57- T cells |
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| Relations |
| BioSample |
SAMN27361365 |
| SRA |
SRX14753676 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6025654_3.barcodes.tsv.gz |
16.3 Kb |
(ftp)(http) |
TSV |
| GSM6025654_3.features.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| GSM6025654_3.matrix.mtx.gz |
22.1 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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