Monocyte Enrichment and RNA Isolation Protocol

For enrichment of monocytes and RNA isolation, the same methodological approach was used at both time points [18 (link)]. In brief, for monocytes enrichment the RosetteSep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, Canada) was used directly after blood sampling. Total RNA was extracted on the same day of blood sampling using Trizol extraction (Invitrogen/Thermo Fisher, Darmstadt, Germany) and purification by the RNeasy Mini Kit (Qiagen, Hilden, Germany). Monocyte enrichment and RNA isolation were performed in the GHS study center by the same personnel for both baseline (BL) and follow up (FU) visit. The integrity of the total RNA was assessed through analysis on an Agilent Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany). Samples with a RNA integrity number (RIN) <7 were excluded. Total RNA was stored at –80°C until further processing; Time of storage was 2–14 month at both time points with the same time span for RNA samples of the same individuals at BL and FU.