Computational protocol: Ionic immune suppression within the tumour microenvironment limits T cell effector function

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Protocol publication

[…] RNA sequencing was performed and analysed as described previously. CD8+62L+ C57BL/6 splenocytes were FACS sorted from 6-8 week old mice in biological triplicate and activated with anti-CD3 and CD28 for 48 hours in IL-2 100 IU mL-1 and cultured in RPMI complete media for an additional 72 hours. Cells were then subjected to ficoll density separation to isolate live cells, placed in complete media without IL-2 in the presence or absence of elevated [K+]e, and either re-stimulated with anti-CD3 and CD28 or kept in complete media for two hours with no stimulation (No stim). Cellular RNA was preserved with RNAlater (Qiagen) and purified with the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). RNA was subsequently used to prepare RNA-seq libraries by using TruSeq SRRNA sample prep kit (FC-122-1001, Illumina) according to the manufacturer’s instructions. The libraries were sequenced for 150bp (paired-end) using a NextSeq500 sequencer (Illumina). Sequence reads from each cDNA library were mapped onto the mouse genome build mm9 by using Tophat, and the mapped data was then processed by Cufflinks. The obtained data was normalized based on RPKM (reads per kilobase exon model per million mapped reads). To define differentially regulated genes, we used a 1.5-fold change difference between treatment groups. Real-time RT-PCR was performed for genes following cDNA generation by reverse transcription (Applied Biosystems) with primers from Applied Biosystems by Prism 7900HT (Applied Biosystems). RNA-sequencing raw data files are deposited at GEO-GSE84996. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Neoplasms
Chemicals Potassium