RNAseq was performed on activated CD8+ T cells sorted from mouse spleenocytes
For library preparation, 6 RNA samples were taken with an initial RNA input of 500ng. KAPA mRNA Capture kit were used for mRNA capture by oligo mag beads, followed by mRNA fragmentation using heat and magnesium. The KAPA RNA Hyper prep kit for Illumina sequencing contains all the buffers and enzymes required for the rapid construction of stranded mRNA-Seq libraries. Next, the cDNA is synthesized using Reverse transcriptase and random hexamers in a first strand synthesis reaction. Subsequently, the cDNA is converted to double stranded cDNA where Uracil is added instead of Thymine and adds dAMP to the 3 ends of the resulting dscDNA. In Adapter ligation, where dsDNA adapters with 3 dTMP overhangs are ligated to library insert fragments followed by library amplification, to amplify library fragments carrying appropriate adapter sequences at both ends using high-fidelity, low-bias PCR. The strand marked with dUTP is not amplified, allowing strand-specific sequencing.