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Sample GSM5469162 Query DataSets for GSM5469162
Status Public on Feb 26, 2024
Title Adult IL-12/18 rep 1 [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-12/18 (bystander activation)
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-12/18 cells
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
BioSample SAMN20362711
SRA SRX11534752

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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