title: RNA-seq of Prdm1 Tcf7 DKO TIM3-SLAMF6+PD1+ CD8+ T cells, 21 dpi LCMV-c13
design: PD1+TIM3-SLAMF6+ CD8+ T cells were sorted from spleens of mice on 21 dpi with LCMV-c13. RNA was isolated using the Zymo Research Direct-zol RNA Microprep Kit. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 10ng of total RNA with a Bioanalyzer RIN score greater than 8.0. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq X Plus using paired end reads extending 150 bases.Basecalls and demultiplexing were performed with Illuminas bcl2fastq software with a maximum of one mismatch in the indexing read.  RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1.  Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.32. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.5.23.