title: GSM4762144: BAL17; Homo sapiens; RNA-Seq
library_construction_protocol: For each patient approximately 20 ml of BAL fluid was obtained, stored at room temperature and processed within 2 hours in a BSL-3 laboratory. An unprocessed aliquot was used for bacterial cul-ture. The BAL fluid was filtered 2 times through a nylon gauze and a 100-μm nylon cell strainer to remove clumps and debris. The supernatant was then washed with PBS 1x and centrifuged. RBCs were lysed with 4 mL of 0.2% NaCl solution (3 minutes, RT) and the reaction was blocked by adding 9 mL of 1.2% NaCl solution. The cells were washed with PBS 1x, re-suspended in RPMI 1640 me-dium supplemented with 5% bovine serum albumin and counted. Cell viability was determined by Trypan blue exclusion. BAL fluids of patients with COVID-19 infection contained a heterogeneous number of cells ranging from 0.83 x106 to 22 x106 of cells. Cells were re-suspended at a concentration of 1 × 106 /ml for single cell analysis. Peripheral blood (PB) from COVID-19 patients and HDs was collected in EDTA-coated tubes. 2 ml of PB was washed once with PBS 1x and the RBCs lysis was performed twice adding 15 mL of 0.2% NaCl solution (3 minutes, RT) and the reaction was blocked by adding 35 mL of 1.2% NaCl solution. The cells were washed with PBS 1x, re-suspended in RPMI 1640 medium supplemented with 5% bovine serum albumin, filtered through a 100-μm nylon cell strainer and counted. Cell viability was determined by Trypan blue exclusion. Cells were re-sus-pended at a concentration of 1 × 106 /ml for single cell analysis For each sample, cells were resuspended in RPMI supplemented with 5% FBS to a final concentration of 1000 cells per ml and processed using the 10x Genomics Chromium Controller and the Chromium NextGEM Single Cell 3′ GEM, Library& Gel Bead kit v3.1 (Pleasanton, California, United States) following the stand-ard manufacturer's instructions. In brief, 10,000 live cells were loaded onto the Chromium controller to recover 4,000 single cell GEMs per inlet uniquely barcoded. After synthesis of cDNA, sequencing libraries were generated. Final 10X library quality was assessed using the Fragment Analyzer High Sensitivity NGS kit (Agilent Technologies, Santa Clara, CA, USA) and then sequenced on the Illu-mina NextSeq500 (Illumina, San Diego CA, USA) generating 75 base pair paired-end reads (28bp read1 and 91bp read2) at a depth of 50,000 reads/cell. Single cell RNA-seq 3' : 28bp R1 and 91 bp R2