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

Purpose: 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 identified following GATK's best practice and only variants unique in one sample were retained. Results. Mapped reads were on average 35,159,030 ± 6,605,340 assigned counts per sample and 1,132 ± 266 mutations.