title: peripheral blood transcriptomics
design: RNA was extracted from 2.5 x 10^5 PBMCs homogenized in 200 mL of Buffer RLT (Qiagen) and then extracted using the Quick-RNA MagBead Kit (Zymo) with DNase digestion. RNA quality was quantitated using Qubit HS RNA assays and assessed using a Fragment Analyzer (Agilent). Library preps were performed using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio) to synthesize full-length cDNA from an input of 10ng of RNA. After a bead-based clean-up to purify the cDNA, the Nextera XT kit was used to create libraries through a process of tagmentation and fragment amplification and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). After another bead-based clean-up, the libraries were normalized, pooled, and sequenced. Libraries were validated by capillary electrophoresis on a Fragment Analyzer (Agilent), pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq6000 or NovaSeq S4 at 100 bp, paired-end read length targeting ~25 million reads per sample. RNA Control spike-ins (Ambion ERCC) were used in a subset of the controls included on each plate. A minimum of two no-template controls were included per library prep plate. Repeated measures from a group of PBMC samples collected from healthy controls and repeated measures of a subset of IMPACC samples were used across library prep and sequencing batches to assess inter-site batch effects throughout the study. Universal Human References controls were included to assess intra-site batch variation. Lastly, a set of reference PBMCs stimulated with TLR7 agonists was included to determine intra-site batch sensitivity.