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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 17, 2024 |
| Title |
donor T1DAL_923889, alefacept treated, 104 wk post-treatment, non-responder (NR), single cell RNA-seq |
| Sample type |
SRA |
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| Source name |
CD3+ CD8+ CD45RA+/- CCR7+/- sorted from PBMC
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| Organism |
Homo sapiens |
| Characteristics |
tissue: CD3+ CD8+ CD45RA+/- CCR7+/- sorted from PBMC
cell line: NA
cell type: non-naive CD8 T cells
maskeddonorid: T1DAL_923889
response (r=responder,_nr=non-responder): NR
cellnumber: 50000
treatment: alefacept
stimulation: None
timepoint: Week 104
lib id: lib48858
visit: 5
genotype: NA
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| Extracted molecule |
total RNA |
| Extraction protocol |
PBMCs were stained with a flow cytometry panel (Figure S12) and sorted into non-naive CD8+ T cells (singlet, live, CD14- CD19- CD56- CD3+ CD8+ CD4-, not CD45RA+ CCR7+) from 12 donors (6 alefacept responders, 6 non-responders, as defined by preservation of C-peptide levels11,58) with new onset T1D at 104 wk (Visit 30) post-treatment with alefacept (T1DAL Trial, ITN) using the FACS Aria Fusion (Becton Dickinson) cell sorter. Cell viability and purity were >90%. Samples were sorted into RPMI with 2% human serum prior to single cell RNA-seq. A single cell suspension was prepared from sorted cells and loaded onto the 10x Chromium Controller (10X Genomics) according to the manufacturer’s protocol, with a target capture of 5,000 cells per channel.
Sequencing libraries were generated using the NextGEM Single Cell 5' v1.1 kit (10x Genomics). Three pools of gene expression and TCR libraries, each from two responders and two non-responders, were generated, with a ratio of 4:1 GEX:TCR. Each pool was run on a NextSeq P2 flowcell on a NextSeq 2000 sequencer (Illumina), with a target depth of 20,000 reads/cell for GEX and 5000 reads/cell for TCR libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
10X Genomics
6_8083_GEX
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| Data processing |
Sequence data were processed from raw reads to gene-barcode count matrices and assembled into TCR sequences using 10x Genomics Cell Ranger v4.0.059. Reads were demultiplexed by sample and library type using index sequence pools. Gene expression reads were aligned to the human genome (GRCh38.91), and reads were collapsed to molecule counts per gene using unique molecular identifiers (UMIs). TCR sequence reads were assembled into consensus contigs, assessed for in-frame and productive sequences, and aligned to the 10x V(D)J reference v. 4.0.0 to determine gene usage. Barcodes were called as cells or background using default settings in CellRanger (10X), which utilizes both the distribution of UMI counts by barcode and expression comparisons of low-UMI-count barcodes to assumed background. Data from all samples were aggregated without normalization prior to downstream analysis.
Assembly: GRCh38.91
Supplementary files format and content: For RNA-seq files: processed data is unfiltered 10X output of single cell RNA-seq in h5 format.
Supplementary files format and content: For TCR-seq files: processed output is in a csv file with information for the assembled TCR chains in each cell output from the 10X Genomics Cell Ranger program.
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| Submission date |
Jul 18, 2023 |
| Last update date |
Jan 17, 2024 |
| Contact name |
Stephanie Osmond |
| E-mail(s) |
sosmond@benaroyaresearch.org
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| Organization name |
Benaroya Research Institute
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| Street address |
1201 9th Ave
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| City |
Seattle, |
| State/province |
WA |
| ZIP/Postal code |
98101 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE237611 |
Interconnected lineage trajectories link conventional and NK-like exhausted CD8+ T cells beneficial in T1D [P362-1] |
| GSE237614 |
Interconnected lineage trajectories link conventional and NK-like exhausted CD8+ T cells beneficial in T1D |
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| Relations |
| BioSample |
SAMN36535371 |
| SRA |
SRX21075556 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7634814_lib48858_raw_feature_bc_matrix.h5 |
25.0 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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