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| Status |
Public on Feb 26, 2024 |
| Title |
IL-2 Neonate [scRNA-seq] |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
cell type: CD8+ T-cells
tissue: Spleen
strain: gBT-I WT
age: Neonate (5 to 7 days)
treatment: IL-2 (control)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Murine cells were isolated from isolated from spleen and 4-6 neonatal tissues were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer. CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads. 2x105 cells were plated per well in round-bottomed 96 well plates. Cells were incubated in RPMI supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, 2-mercaptoethanol (RP-10) and 10 ng/ml recombinant human IL-2 (Thermo Fisher Scientific) for 18-22 hours. For bystander-activated samples, media was additionally supplemented with recombinant murine IL-12p70 (Thermo Fisher Scientific) and recombinant murine IL-18 (Thermo Fisher Scientific) at 1 ng/ml. 1.5 mg/ml Brefeldin A (Millipore Sigma; St. Louis, MO) was added to the cells for the final 4 hours of incubation prior to antibody labeling for flow cytometry analysis. Live Va2+CD8+ cells were sorted into 0.04% BSA in PBS.
Cells were counted and 3-8k cells per sample were loaded in the Chromium controller for the formation of gel bead-in-emulsions (GEMs) following the manufacturer's instructions (10X Genomics). The single-cell RNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit v2 following the accompanied protocol and were sequenced on Illumina NextSeq500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
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| Data processing |
The raw FASTQ data was processed using Cell Ranger v3.0.1
Count matrixes were filtered using Seurat v2.3.3 to remove cell barcodes with less than 200 genes and with more than 5% mitochondrial counts
Seurat v2.2.3 was used to normalize, scale, regress out variation in UMI counts and mitochondrial content, perform dimensionality reduction and generate UMAP projections.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files; containing cell barcodes, genes, and matricies which can be combined to form a Barcodes X Features sparse count matrix for each sample.
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| Submission date |
Nov 21, 2022 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Andrew Grimson |
| Organization name |
Cornell University
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| Department |
Molecular Biology and Genetics
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| Street address |
445 Biotech Bldg
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| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14850 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE180732 |
Gene regulatory basis of bystander activation in CD8+ T cells |
| GSE218456 |
Gene regulatory basis of bystander activation in CD8+ T cells (scRNA-seq) |
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| Relations |
| BioSample |
SAMN31818474 |
| SRA |
SRX18329007 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6745550_IL2_Neonate_barcodes.tsv.gz |
23.1 Kb |
(ftp)(http) |
TSV |
| GSM6745550_IL2_Neonate_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM6745550_IL2_Neonate_matrix.mtx.gz |
27.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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