RNASeq analysis of solid tumours from CT26 syngeneic mice to study the role of T-cells and tumour intrinsic pathways (STING/IFN) for driving ceralasertib (AZD6738) efficacy
RNASeq analysis of solid tumours from CT26 syngeneic mice to study the role of T-cells and tumour intrinsic pathways (STING/IFN) for driving ceralasertib (AZD6738) efficacy
CT26 and A20, BALB/c mice were subcutaneously implanted into the right flank with 5x105 and 1x107 cells respectively. Tumors were measured using digital calipers measured 3x a week until the end of study or when tumors reached the ethical limit of 1500- 2000mm3 using calipers.  CT26 tumor tissues were isolated and total RNA was extracted using the RNeasy 96 QIAcube HT Kit (Qiagen; C/N 74171) according to manufactuers guidelines with a DNase digest included. RNA concentration was determined by Qubit Flex Fluorometer (Invitrogen), RNA purity was determined using a NanoDrop Eight (Thermo Scientific) and RNA integrity was measured using a 4200 Tapestation (Agilent). All samples had a RINe of >7.0. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; #E7760L) as per manufactuer's guidelines with 1000ng of RNA input. Ribosomal RNA was removed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs; E7490L) Libraries were subjected to 9 cycles of PCR with unique dual indexes (New England BioLabs; E6440L). Libraries were subsequently quantified by Qubit Flex Fluorometer (Invitrogen), and library sizes were determined by 4200 Tapestation (Agilent) and pooled equimolar.