RNA-seq of activated CD 8 T Cell
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) and quantified using Qubit (Thermo Fisher Scientific). For library prep, SMART-Seq v4 Ultra Low Input Kit (Clontech) was used for full-length cDNA synthesis and amplification; Illumina Nextera XT libraries were prepared via transposase-based fragmentation and indexed via limited-cycle PCR. Libraries were validated on Agilent TapeStation and quantified using Qubit 3.0 and qPCR (KAPA Biosystems). Libraries were multiplexed, clustered on an Illumina NovaSeq flowcell, and sequenced in 2x150 bp paired-end mode. Base calling was performed using NovaSeq Control Software (NCS); raw .bcl files were converted to FASTQ and demultiplexed using Illumina bcl2fastq 2.20 (allowing one index mismatch).