Co_360M3
Homo sapiens
Protocols: For cell isolation and purification from individuals, peripheral blood mononuclear cells (PBMCs) were collected from severe COVID-19 patients and healthy controls, cryopreserved, and stored in liquid nitrogen. The PBMCs were then thawed, revived, and CD14+ monocytes were isolated using magnetic bead separation. This process involved resuspension in binding buffer, addition of CD14 magnetic beads, incubation, washing, and magnetic separation to collect the CD14+ monocytes For RNA purification, total RNA was extracted from the isolated CD14+ monocytes using the Qiagen RNeasy kit following the manufacturer's instructions. This process included cell lysis, addition of ethanol to adjust binding conditions, loading onto a silica membrane column, washing to remove impurities, and elution of RNA. The quality and quantity of RNA were assessed with a NanoDrop spectrophotometer and Agilent Bioanalyzer, ensuring suitability for uploading to a public repository. RNA was purified from  CD14+ monocytes. PolyA selected strand-specific libraries were generated with 800ug of input RNA using the Illumina Stranded mRNA Prep (Illumina document 1000000124518 v03). Samples were sized, quantified and validated on a Bioanalyzer.