GSM5642555: Healthy control blood rep2 (GEX); Homo sapiens; RNA-Seq
Blood was collected bedside via standarad venipuncture into EDTA (lavender top) or sodium heparin (green top) vacutainer tubes. Blood was maintainted and transferred to the laboratory at room temperature for processing. Plasma from whole blood was isolated via centrifugation at 400 x g for 10 min at 4˚C. Untouched circulating leukocytes were isolated using the EasySep™ RBC Depletion Reagent (StemCell Technologies; Cat. #18170). Endotractheal aspirates (ETA) were collected bedside and transported to the laboratory at room temperature where it was then placed at 4˚C for processing in BSL3 containment. ETA was mixed 1:1 with a 50mM EDTA solution (final concentration 25 mM EDTA) in custom RPMI-1640 media deficient in biotin, L-glutamine, phenol red, riboflavin, and sodium bicarbonate (defRPMI-1640), with 3% newborn calf serum (NBCS) and mechanically dissociated using a syringe to liberate leukocytes from mucins and other respiratory secretions. ETA cells underwent an additional mechanical dissociation step using 1-3 mL of a 10 mM EDTA in defRPMI-1640 + 3% NBCS and a P1000 pipettor. Cells were then washed with 10 mL defRPMI-1640 + 3% NBCS, passed through a 70 µm nylon strainer, and pelleted through a 2 mL 100% NBCS layer. Leukocytes from whole blood and ETA samples were incubated with oligo-conjugated Ig-A/D/G/M for 10 min at 4˚C followed by the addition of Human TruStain FcX™ (BioLegend; Cat #422302) and 10 min incubation at RT. Cells were then surface stained with oligo-conjugated monoclonal antibody panel (total 89 antibodies; Extended Data Table 5) for 30 min at 4˚C, followed by two washes in def-RPMI-1640/0.04% BSA. Cells were resuspended at a concentration of 1200-1500 cells/µL in def-RPMI-1640/0.04% BSA and passed through a 20 (blood) or 40 µm (ETA) cell strainer before loading onto a Chromium Controller (10X Genomics, Pleasanton, CA). Gene expression (GEX) and antibody-derived tag (ADT) libraries were generated using the Chromium Single Cell 5′ Library & Gel Bead Kit v1.1 with feature barcoding following the manufacturer's instructions (https://support.10xgenomics.com/single-cell-vdj/index/doc/user-guide-chromium-single-cell-vdj-reagent-kits-user-guide-v11-chemistry-with-feature-barcoding-technology-for-cell-surface-protein).
GEO Accession
GSM5642555