GSM4983712: 10084-AI11-NAE-1; Homo sapiens; RNA-Seq
Activated antigen specific CD8 T cells (defined as CD69+/CD137+) were first disaggregated into a single-cell suspension and placed in 96 well plate containing lysis buffer containing a ribonuclease inhibitor. Single cell RNA-sequencing methodology was adapted from two previously described protocols, Smart-seq2 and MARS-Seq. Following the first round PCR, fragmentation and end-repair were performed using the NEBNext Ultra II FS kit, as per the manufacturer's instructions. Adaptors were ligated to amplicons using the NEBNext Ultra II FS kit, as per the manufacturer's instructions. A second round PCR was undertaken using GoTaq and linker primer 1. Following this, a third round PCR was conducted with barcoded linker primer 2. PCR products were purified and pooled for next-generation sequencing. The Kapa library quantitation kit  was used to quantify the libraries prior to sequencing on the Illumina MiSeq sequencer using the 600V3 kit (Illumina Inc.), as per the manufacturer's instructions.