title: GSM7432061: P18_Bulk_CD8; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were isolated and FACS sorted based on CD4, CD8, and PD-1 expression. CD8 T cells were directly sorted into 96 well plates prepared with 1X Reaction Buffer consisting of lysis buffer and RNase Inhibitor for low input RNA sequencing (Takara Bio, USA). Plates were spun down and immediately stored on dry ice or at -80°C until further processing. Sample plates containing lysed T cells were subjected to cDNA library preparation using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio, USA) followed by sequencing library preparation using the Nextera XT DNA Library Preparation Kit (Illumina) as per manufacturer's instructions with minor modifications. Briefly, full-length cDNA was generated by reverse-transcription, template-switching reaction and PCR pre-amplification of polyadenylated mRNA as previously described. cDNA libraries were quantified using the Qubit dsDNA High Sensitivity Kit and quality was assessed on a Bioanalyzer using DNA High Sensitivity chips (Agilent). Double-stranded cDNA was subjected to fragmentation and PCR-based addition of Illumina barcoded sequencing adapters at both fragment ends. Sequencing library quantity and quality was assessed as described above.