title: RNA-seq of KLRG1+ effector CD8 T cells d8 Arm, WT
design: 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 GRCm39.113 primary assembly with STAR version 2.7.11b.  Gene counts were derived from the number of uniquely aligned unambiguous fragments by Subread:featureCount version 2.0.8. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.10.0.  Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected.  The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 5.04.