|
| Status |
Public on Feb 26, 2024 |
| Title |
Adult IL-2 rep 6 [RNA-seq] |
| Sample type |
SRA |
| |
|
| Source name |
Splenic CD8+ T-cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Splenic CD8+ T-cells
age: Adult (2 to 4 months)
treatment: IL-2 (control)
genotype: gBT-I WT
|
| Treatment protocol |
Total of 200,000 cells were plated per well in round-bottomed 96 well plates and 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.
|
| Growth protocol |
Spleens were harvested from TCR Tg gBT-I adult, neonate or Lin28b Tg adult mice, and 4-6 neonatal spleens were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer and CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads (Miltenyi Biotec) according to the manufacturer’s instructions.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated with Trizol, with an extra chloroform extraction to remove residual phenol and addition of glyco-blue as a carrier to promote RNA precipitation.
RNA-seq libraries were prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), with initial polyA+ isolation, by the Transcriptional Regulation and Expression Facility at Cornell University.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
RNA-seq analysis of Adult IL-2 cells
read_counts.tsv
|
| Data processing |
Illumina pipeline software was used for base calling.
Raw reads were trimmed to remove adapters using cutadapt and were mapped to the mouse genome (mm10) using tophat (--no-novel-juncs --library-type fr-firststrand).
The reads mapping to each gene were counted using featureCounts (-s 0 -Q 50).
The differential expression analysis of RNA-seq was performed using edgeR.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-separated count file, containing counts of RNA-seq reads mapping to exons
Supplementary_files_format_and_content: edgeR output files; tab-separated edgeR output files for RNA-seq data
|
| |
|
| Submission date |
Jul 23, 2021 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Andrew Grimson |
| Organization name |
Cornell University
|
| Department |
Molecular Biology and Genetics
|
| Street address |
445 Biotech Bldg
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14850 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE180730 |
Gene regulatory basis of bystander activation in CD8+ T cells (RNA-seq) |
| GSE180732 |
Gene regulatory basis of bystander activation in CD8+ T cells |
|
| Relations |
| BioSample |
SAMN20362724 |
| SRA |
SRX11534751 |
