Bronchoscopy with bronchoalveolar lavage (BAL) was performed under conscious sedation (Bailey, et al., 2018; Fini, et al., 2017; Gaydos, et al., 2016). Briefly, the bronchoscope was wedged into a subsegment of either the right middle lobe or lingula, followed by instillation of 50-mL aliquots of sterile, room temperature 0.9% saline. Each 50-mL aliquot (up to four total) was immediately hand aspirated back into the syringe, then transferred to sterile 50-mL conical tubes, whereupon they were transported to the laboratory. The first aliquot of BAL was not used in these experiments. Second and subsequent BAL aliquots were combined, then centrifuged to separate cellular and acellular components. Cell counts were performed, and ≥ 95% BAL cell viability was determined for all subjects via trypan blue exclusion. Differentials were assessed after Diff-Quik staining by a single blinded observer. A proportion of BAL cells were snap frozen for RNA applications, while the remainder were used in ex vivo cell culture experiments. RNA library creation and sequencing were performed at the Anschutz Medical Campus Genomics and Microarray Core in two batches: Batch 1 (TruSeq Stranded mRNA Library Prep Kit and Illumina HiSeq4000; Illumina Inc., San Diego, CA) and Batch 2 (Illumina TruSeq Stranded Total RNA kit, HiSeq2500) using manufacturer directions. Note that batch is provided as a variable in the sample metadata.
GEO Accession
GSM4697494