Tempus rna blood tubes

Peripheral blood was collected from study participants into Tempus RNA Blood Tubes (ThermoFisher, #4451893). As soon as possible after blood collection, tubes were stored at −80°C until RNA extraction. The MagMax protocol for Stabilized Blood Tubes RNA Isolation Kit (Ambion, Life Technologies) was used to extract total RNA from 3 mL of collected peripheral blood, following the manufacturer’s instructions. Briefly, frozen blood tubes were thawed to 4°C prior to RNA extraction. In a biosafety level 2+ (BSL2+) cabinet using proper personal protective equipment (PPE), the blood was first diluted with 1X PBS, then pelleted and washed at 4°C. The washed and re-pelleted RNA was then dissolved in Resuspension Solution followed by a dual proteinase and DNase treatment. Next, the RNA was purified through a series of magnetic bead-based washes and eluted in 80 μl of Elution Buffer as described in the user manual, prior to downstream quantification and library preparation steps. In addition, these modifications to the protocol were followed during extraction: 1) all processing was performed inside a BSL2+ cabinet until resuspension solution was added to the samples, 2) samples were kept on ice throughout the protocol, 3) wash 1 buffer, wash 2 buffer, and isopropanol were kept on ice and used cold, 4) samples were shaken on a vortex adaptor at a setting of 4.

Patients presenting to the Mount Sinai Health System (MSHS) between April and June of 2020 were enrolled through daily manual review of new hospitalizations for COVID-19. Blood collection was performed in conjunction with routine clinical blood draws throughout the participants’ hospital stay. Research specimens were brought to biosafety level 2 plus facilities for accessioning, processing, and storage of serum, plasma, whole-blood, and peripheral blood mononuclear cells (PBMCs). The whole blood utilized for RNA sequencing (RNA-seq) was collected in Tempus RNA Blood Tubes (ThermoFisher, #4342792). As soon as possible after blood collection, tubes were shaken and stored at −80°C. Blood used for Olink and enzyme-linked immunosorbent assays (ELISA) were collected in SST tubes (Becton Dickinson #367985) and blood used for whole genome sequencing (WGS) in CPT Vacutainer tubes (Becton Dickinson #362761). Blood in SST tubes was centrifuged to extract serum that was then aliquoted and stored at −80°C in Cryovials (Crystalgen #19335–6SPR). Blood from CPT tubes was aliquoted for WGS and stored at −80°C in Cryovials (Crystalgen #19335–6SPR).