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Status |
Public on Feb 01, 2021 |
Title |
Tmem_pooled |
Sample type |
SRA |
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Source name |
CD8 T cells
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Organism |
Mus musculus |
Characteristics |
background strain: C57BL/6 treatment: LCMV Armstrong infection
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Treatment protocol |
Live CD8+ congenic (CD45.1+ or CD45.1/2+) LCMV-GP33-tetramer+ P14 cells were FACS-sorted (1.18–2.4 x104 cells), used to generate single-cell gel-beads in emulsion, and counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific) prior to library preparation
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Growth protocol |
LCMV-specific P14 cells were sorted from mice infected with LCMV-Arm or LCMV-Cl13, as well as mice that received donor P14 TEX or TMEM
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Extracted molecule |
total RNA |
Extraction protocol |
Cells were loaded onto the Chromium Controller (10x Genomics) for a target recovery of 5,000 single cells. The scRNAseq libraries were generated using Chromium Single Cell 3′ Library and Gel Bead Kit v2 (10x Genomics) according to the manufacturer’s protocol. Briefly, emulsions were created and, after reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR (12 cycles). Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR (8 cycles). Samples were pooled and quantified using a KAPA Library Quantification Kit (Kapa Biosystems) prior to sequencing
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
NextSeq 550 |
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Description |
Tmem_pooled
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Data processing |
Sample demultiplexing, barcode processing, alignment, filtering, unique molecular identifier (UMI) counting and aggregating sequencing runs were performed using Cell Ranger software v.2.1.0 (10x Genomics). Downstream analysis was performed using Seurat v2.3 (Butler, et. al. 2018). Briefly, individual library outputs from the Cell Ranger pipeline were loaded, and cells with fewer than 200 detected genes were excluded from downstream analysis. Genes detected in fewer than three cells across the dataset were also excluded. Raw UMI counts were normalized by total expression in the corresponding cell and multiplied by a scaling factor of 10,000 to give UMI count per million total counts and then log-transformed Variable genes were selected based on average expression and dispersion. Linear dimensional reduction (PCA) was performed using variable genes and the first 7 principal components were determined to be significant using the jackstraw method. tSNE was performed on these significant principle components using default parameters for 1,000 iterations for visualization in two dimensions. Unsupervised clustering was performed using a shared nearest neighbor modularity optimization-based algorithm. Optimal number of clusters was chosen using Identifying K mAjor cell Population groups (IKAP) algorithm Differential expression analysis was performed between each cluster and all other cells using a Wilcoxon rank sum test. Marker genes were identified by the Seurat function FindAllMarkers,Scaled expression data of these marker genes were used for creating the heatmaps. Normalized data are shown in the form of feature plots or violin plots. Trajectory analysis was performed with Monocle v.2 conducted using Monocle 2 v2.10.1 . Genome_build: GRCm38/mm10 Supplementary_files_format_and_content: Filtered gene-barcode matrices containing only cellular barcodes
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Submission date |
May 12, 2020 |
Last update date |
Feb 02, 2021 |
Contact name |
E JOHN WHERRY |
Organization name |
University of Pennsylvania
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Department |
Systems Pharmacology and Translational Therapeutics,Institute for Immunology
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Street address |
421 Curie Blvd,354 BRB II/III
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City |
Philadelphia |
State/province |
PA |
ZIP/Postal code |
19104 |
Country |
USA |
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Platform ID |
GPL21626 |
Series (2) |
GSE150369 |
Transcriptional landscape of three exhausted CD8 T cell subsets |
GSE150370 |
Exhausted T cells recovered from chronic antigen stimulation |
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Relations |
BioSample |
SAMN14896551 |
SRA |
SRX8329792 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4547620_Tmem_barcodes.tsv.gz |
16.1 Kb |
(ftp)(http) |
TSV |
GSM4547620_Tmem_genes.tsv.gz |
217.2 Kb |
(ftp)(http) |
TSV |
GSM4547620_Tmem_matrix.mtx.gz |
9.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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