scRNAseq of Antigen Presenting Cells in severe COVID-19 patients

We isolated PBMC from healthy, moderate ( Oxygen supply < 10L/min), and severe (Oxygen supply >= 10L/min) COVID-19 patients after their admission to Intensive Care Units (ICU), at two timepoints (Day-1 and Day-4); and performed both CD14+ Monocyte enrichment followed by a Pan-DC kit to retrieve all Antigen Presenting Cell (APC) subsets from these age-matched patients. We performed single cell RNA sequencing using 10X technology on the single cell suspensions and constracted a high-resolution map of 81,643 Antigen Presenting Cells (APC) from the three COVID-19 severity groups. We were able to retrieve all the known six APC subsets and deciphered the altered pathways and ati-viral mechanisms, correlated with the disease severity. After FICOLL (GE Healthcare and Lymphoprep StemCell) gradient centrifugation, total PBMC were enriched in CD14 monocytes using the human CD14 microbeads (Miltenyi Biotec) for positive magnetic selection according to the manufacturer’s instructions. The negative fraction remaining after the positive selection of CD14 cells was used for pan-DC enrichment employing the EasySep human pan-DC enrichment kit (StemCell Technologies). Total pan-DC were resuspended with 20,000 CD14 cells and sent for sequencing. Cell suspensions were subjected to Gel Bead Emulsion using Chromium 10x Genomics controller according to manufacturer guidelines. Both library preparation and sequencing were performed accroding to manufacturer guidelines.