NextSeq 550 paired end sequencing; RNA-seq analysis of CD8 tumour infiltrating lymphocytes from GFP-Egr2 Knockin and hCD2-Cre Egr2loxP/loxP Egr3-/- mice
RNA-seq analysis of CD8 tumour infiltrating lymphocytes from GFP-Egr2 Knockin and hCD2-Cre Egr2loxP/loxP Egr3-/- mice
CD3+CD8+CD44highGFPEgr2high cells were isolated from tumour cell suspensions from Egr2 Kin mice by FACS Tumour cell suspensions were prepared from solid tumors by enzymatic digestion in HBSS (Life Technologies) containing 1 mg/ml collagenase, 0.1 mg/ml DNAse I, and 2.5 U/ml of hyaluronidase (all from Sigma-Aldrich) with constant stirring for 45 minutes at 37oC. A total of 1x10^5 MC38 tumour cells were injected subcutaneously into GFP-Egr2 knockin (Egr2 Kin) and hCD2-Cre Egr2loxP/loxP Egr3-/- (Egr2/3 DKO) mice. Tumours were harvested 14 days later RNA was isolated and purified using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. cDNA libraries were generated using the NEBNextÂ® Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs, E6420), which uses SMART (Switching Mechanism at 5' End of RNA Template) technology and produces full-length PCR amplified cDNA starting from small amounts of total RNA. The integrity and quantity of the amplified cDNA was verified on the Agilent Bioanalyser using the High Sensitivity DNA kit. 200pg of cDNA was then tagmented using the Nextera XT protocol (Illumina, FC-131-1024) and amplified with custom primers containing P5/P7 sequences along with a 6bp sample specific index to generate the sequencing libraries.