RNA-seq Analysis of Disease Severity

RNA was extracted from 300K cells using the Zymo Direct-zol miniprep kit (Cat# R2051) and RNA quality assessed using the Agilent DNA tape screen assay. The RNA Integrity Number (RIN) scores for all samples were > 7.0. Total RNA libraries were generated using the NuGEN Ovation® SoLo RNA-Seq Library Preparation Kit (Cat# 0500–96). Libraries were sequenced using Illumina NovaSeq 6000 instrument with S4 flow cell and 150 base pair paired-end reads.FASTQ files were generated from the NovaSeq BCL outputs and quality was assessed with FASTQC²⁹ . Differentially expressed genes were identified between subjects with different disease severity using the limma package and voom to model variance^{30 (link)}.