Daan Pieren

127 Regulatory KIR+RA+ CD8+ T cells accumulate with age Library preparation FACS-sorted T NAIVE , T EMRA , KIR + RA + , and NKG2A + RA + T-cell subsets of six healthy donors were centrifuged at 485 g for 15 minutes and lysed in buffer RLT (Qiagen) containing 1% β -mercaptoethanol. Total RNA was extracted from the cell lysates by using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA integrity was assessed by using the RNA 6000 Pico kit (Agilent Technologies) on a 2100 BioAnalyzer (Agilent Technologies). All RNA Integrity Number (RIN) scores were >7.0. cDNA synthesis and amplification was performed by using the SMART-Seq® v4 Ultra® Low Input RNA kit for sequencing (Takara) and AMPure XP beads (Beckman Coulter) were used to purify the samples. Proper size distribution of the fragments acquired was verified using the High Sensitivity DNA kit (Agilent Technologies). Next, 1 ng of cDNA was used to prepare a Nextera XT DNA library according to the manufacturer’s instruction (Illumina). Libraries were subsequently validated for fragment size using QIAxcel DNA Screening Kit (Qiagen) and quantified using RT-qPCR with a KAPA Library Quantification kit (KK4824, Roche/KAPA Biosystems). 23 libraries were pooled at equimolar concentrations and sequenced using the Illumina NextSeq 500/550 High Output Kit v2.5 (single-end, 75-cycles). Basecalling and demultiplexing was performed using bcl2fastq2 Conversion Software v2.20, and demultiplexed FASTQ files which were generated based on sample-specific barcodes (>14 million reads/sample). RNA sequencing analysis We used an in-house pipeline to analyze the RNA sequencing data. First, reads were mapped to the human reference genome (GRCh38, release 12) using STAR (version 2.6.0) [32]. The number of mapped reads were counted for each gene and compiled into an expression matrix using featureCounts (version 1.6.1) [33]. The count table was used for statistical analysis and identification of Differentially expressed genes (DEG) using DESeq2 (v1.1) [34]. Genes were considered significantly different with a false discovery rate (FDR) < 0.1. The RNA-seq data discussed in this article will be deposited and made accessible in the National Center for Biotechnical Information Gene Expression Omnibus 23 upon acceptation for publication. Ingenuity Pathway Analysis (IPA, Qiagen) was used to investigate the relationships between selected pairs of T-cell subsets. Gene expression profiles of T EMRA , KIR + RA + , and NKG2A + RA + T-cell subsets were first separately compared to the gene expression profile of T NAIVE cells. These comparisons were then aligned in a heat map to identify shared and unique molecular and cellular functions of T EMRA , KIR + RA + , and NKG2A + RA + T cells. For this enrichment analysis, an absolute z-score of greater than 2.0, a False 5