Illumina NovaSeq 6000 paired end sequencing; RNA-seq of RBC depleted whole blood from COVID-19 patients and controls
RNA-seq of RBC depleted whole blood from COVID-19 patients and controls
Whole human peripheral blood was collected into sterile vacutainer tubes containing EDTA for single cell RNA sequencing, tubes were centrifuged at 300 g for 5 min at room temperature and plasma was collected.  To fully capture peripheral blood cell heterogeneity, we analyzed fresh samples without cell sorting or freezing and without Ficoll enrichment, minimizing time of incubation and processing. Sample preparation was done at room temperature. After red cell lysis, single-cell suspensions were loaded onto a Chromium Single Cell Chip (10x Genomics) according to the manufacturer's instructions for co-encapsulation with barcoded Gel Beads at a target capture rate of ~7000 individual cells per sample. To analyze neutrophils, we added RNase inhibitor (RNAse OUT Recombinant Ribonuclease Inhibitor Invitrogen, 40U/mL) into the loading buffer. Captured mRNAs were barcoded during cDNA synthesis using the Chromium Single Cell 3' Solution v3 (10x Genomics) according to the manufacturer's instructions.  Of note, we increased the PCR cycles by two during cDNA amplification. All samples (at Day 0 and Day 10) were processed simultaneously with the Chromium Controller (10x Genomics) and the resulting libraries were prepared in parallel in a single batch.