RNA-Seq of peripheral blood T cells from a human Glioblastoma patient: Sample 99_BT228BTeff-PD1plus_N706-N506
T cells from healthy controls and GBM patients were sorted into RNA lysis buffer for RNA sequencing as described previously. Briefly, RNA was extracted using the NucleoSpin RNA XS Kit (Macherey-Nagel) according to the manufacturer’s instructions. cDNA synthesis and amplification were performed using SMARTer Ultra Low Input RNA for the Illumina Sequencing High Volume Kit (Clontech) according to the manufacturer’s instructions, with the following for LD amplification: 98°C for 10 seconds, 65°C for 30 seconds, and 68°C for 3 minutes 18 times and 72°C for 10 minutes. cDNA was normalized to an input of 0.5 ng total after quantification using Picogreen (Invitrogen) for preparing sequencing libraries. Paired-end sequencing libraries were prepared using the Nextera XT DNA sample Prep Kit (Illumina) according to the manufacturer’s instructions. Libraries were pooled in an equimolar ratio and sequenced on a HiSeq 2500 sequencer with 200 cycles per lane (Illumina).