GSM5821040: Healthy Control 2 mRNA; Homo sapiens; RNA-Seq
Four healthy control (HC) subjects and three subjects with CF underwent research bronchoscopy as previously described (20, 21). Briefly, after local anesthesia to the posterior pharynx and intravenous conscious sedation, a flexible fiberoptic bronchoscope was inserted transorally and passed through the vocal cords into the trachea. The bronchoscope was sequentially wedged into tertiary bronchi in the right upper lobe (RUL), right middle lobe (RML) and right lower lobe (RLL), saline was instilled, and BALF was collected. An aliquot of 10 mL of BALF from the RML was used for the studies outlined here. All subjects provided written informed consent, and this study was approved by the Institutional Review Board of Dartmouth-Hitchcock Medical Center (protocol #22781). Sample processing and library construction were performed immediately after lavage. 1X phosphate-buffered saline (PBS) containing 0.5 mM EDTA, and 0.1% bovine serum albumin (BSA) was added to the BALF. Samples were filtered through 100-micron cell strainers and centrifugated at 300 x g for five minutes at 4 °C and supernatants were discarded. TotalSeq antibodies were added at concentrations recommended by the manufacturer and stained for 30 min on ice (22), details in online supplement. After staining, chilled Hank's balanced salt solution (HBSS) supplemented with 0.5% BSA was used to wash the sample. Samples were again filtered through 100-micron cell strainers and spun as above. Supernatants were aspirated, and pellets were resuspended with HBSS plus 0.5% BSA at an approximate concentration of 7.5 × 105 cells/mL. Cell quality and viability of the samples were assessed with a Cellometer K2 (Nexcellom Bioscience). All samples had viability >80%. Single cells were then processed using the Chromium Next GEM Single Cell 3' Platform (10X Genomics, Pleasanton, CA). Approximately 30,000 cells were loaded on each channel with an average recovery rate of 24,000 cells. Libraries were sequenced on NextSeq 500/550 with an average sequencing depth of 50,000 reads/cell (Illumina, San Diego, CA).