Inherited PD-1 deficiency: RNA sequencing (RNASeq) of sorted double-negative T cells and monocytes

Double-negative (DN) αβ T cells (αβTCR+CD3+CD4-CD8-CD19-) and monocytes (CD3-CD14+/dimCD19-CD56-) were sorted from cryopreserved peripheral blood mononuclear cells (PBMCs). Total RNA was subjected to the library preparation using the SMART-Seq v4 Ultra Low Input RNA Kit with 12-16 cycles of amplification. The cDNA was further amplified using the KAPA Hyper Prep Kit with additional eight PCR cycles. Samples were barcoded and analyzed on a HiSeq 4000 in paired-end 2x100 bp mode, with the HiSeq 3000/4000 SBS Kit. The FASTQ files were first inspected with fastqc to ensure that the raw data were of high quality. The FASTQ reads of each sample were then aligned to the GENCODE human reference genome GRCh38.p13 with STAR aligner ver. 2.7, and the alignment quality of each BAM file was evaluated with RSeQC. Reads were quantified with featureCounts ver. 1.6, to generate gene-level feature counts from the read alignment. Reads were aggregated by gene symbol. Genes on the X or Y chromosome were excluded from the differential expression analysis.