GSM9669498: M14 Pool 2; Mus musculus; RNA-Seq
On day 7, mice were euthanized by isoflurane inhalation and decapitation. The turbinates were then removed from the nasal cavity and collected in freshly prepared oxygenated Ringer's solution containing 115 mM NaCl, 5 mM KCl, 2 mM MgCl2 hexahydrate, 2 mM CaCl2 dihydrate, 25 mM NaHCO3, 10 mM HEPES, and 10 mM glucose with a pH of 7.4 at 4°C. The olfactory epithelium was dissected from the turbinates, minced with fine forceps, then dissociated for 45 minutes at 37°C with tissue dissociation cocktail: dispase II (2 mg/mL), Collagenase Type 1 (2 mg/mL), Papain (0.25 mg/mL), and DNase (2 mg/mL) in Ringer's solution). The reaction was terminated with Ringer's-FBS (FBS-10%) and the cells were triturated, filtered, and centrifuged. The cell suspension was filtered once more through a 40-micrometer strainer, centrifuged, and resuspended in 500 μl of Ringer's solution. Dissociated single cell suspensions were used for single cell capture via 10x Genomics platform. Tissue harvesting, dissociations, and captures were repeated on subsequent days to perform the experiment on 8 total animals (1 male and 1 female for 4 conditions) in a sequencing experiment. Male and female mice were processed on subsequent days/batches. This experiment was performed twice for a total of 16 animals (8 male, 8 female, 4 per condition). In addition to the 16 exposed mice, we also included 2 male mice taken directly from the cage (no exposure) which were processed similarly. These mice (A1 and B1) in the data provided here were used only for cell clustering and not used for differential gene analysis. Samples were barcoded using the 10x Genomics CellPlex cell hashing protocol per manufacturer's recommendations (10x Genomics, Pleasanton, CA). Barcoded samples were pooled and captured on the 10x Genomics Chromium platform per manufacturer's instructions. Pooled samples from 4 MOEs (one male or female from each exposure condition) were captured across 2 independent captures to facilitate sufficient cells per animal/sample and decrease batch effects. Total RNA was isolated, cDNA libraries constructed (single cell 3' v3 protocol, 10x Genomics), and libraries were sequenced on the Illumina NovaSeq6000 (Illumina, San Diego, CA) for a target of 100,000 reads per cell for gene expression + 5000 reads per cell for the cell multiplexing libraries.