GSE196787 Nucleotide metabolism in cancer cells fuels a UDP-driven macrophage cross-talk promoting immunosuppression and immunotherapy resistance [RNAseq_Panc02_sgNT_sgCda_invitro]

Contributor : Massimiliano MazzoneSeries Type : Expression profiling by high throughput sequencingOrganism : Mus musculusPurpose: To study the effects of CDA depletion on PancO2 cell line. Here, we used RNA sequencing to understand if sgCda can alter cancer cell immunogenicity by looking at mutational burden.Methods: PancO2 cancer cells were transduced with a doxycycline-inducible Cas9 nuclease (Edit-R Inducible Lentiviral Cas9, Dharmacom) and selected with blasticidin (Bio-Connect). Cells expressing the doxycycline-inducible Cas9 nuclease were transduced with a target specific gRNA for CDA and a control non-targeting NT gRNA, and selected with puromycin (Sigma-Aldrich). After selection, cells were treated for seven days with or without doxycycline (2.5ug/mL, Sigma-Aldrich) to induce Cas9 expression and grown for seven more days without doxycycline before being used. Then RNA was isolated using TRIzol (Life Technologies). Starting from total RNA, poly-adenylated fragments were isolated, reverse transcribed and converted into indexed sequencing libraries using the KAPA stranded mRNA-seq kit (Sopachem, Eke, Belgium). The first 50 bases of these libraries were sequenced on a HiSeq 2500 system (Illumina, San Diego, CA). These raw reads were mapped after removal of the sequencing adapters to the reference transcriptome and genome (GRCm38/mm10) using the Bowtie TopHat pipeline (Langmead and Salzberg, 2012). Mapped reads were assigned to ensemble gene IDs by HTSeq. Variants were i...
Source: GEO: Gene Expression Omnibus - Category: Genetics & Stem Cells Tags: Expression profiling by high throughput sequencing Mus musculus Source Type: research