Bulk RNA-seq of PBMC Samples

For bulk RNA sequencing of PBMC samples from the independent Singapore cohort (see above), we performed reverse transcription and amplification using a standard protocol^{83 (link)}. Briefly, we synthesized cDNA from 2 ng of purified total RNA using modified oligo(dT) primers, and cDNAs were further amplified. The quantity and integrity of cDNA were assessed using the DNA High Sensitivity Reagent Kit (Perkin Elmer: LabChip GX). Subsequently, pooled cDNA libraries were prepared (250 pg of cDNA per sample, Illumina Nextera XT kit, Cat no. FC-131-1096) with dual indices for de-multiplexing. The libraries were quantified using qPCR (Kapa Biosystems) to ascertain the loading concentration. Samples were subjected to an indexed PE sequencing run of 2 × 151 cycles on an Illumina HiSeq 4000. We mapped paired-end reads to human genome build GRCh38 using the STAR aligner⁸⁴ and counted reads mapped to genes using featureCounts⁸⁵ and GENCODE v31 gene annotations^{86 (link)}. We quantified gene expression as log2-transformed reads per exonic kilobasepair per million mapped reads (log2RPKM) using the edgeR Bioconductor package⁸⁷ . Gene expression estimates for SOCS3 were then analyzed for association with disease severity (D.K. et al.⁴⁹ , unpublished observations).