GSM5608796: Mouse 3: single-cell RNA sequencing of the mouse main olfactory epithelium; Mus musculus; RNA-Seq
Mice were euthanized with intraperitoneal injection of pentobarbital (150mg/Kg) and their olfactory epithelia were immediately extracted and processed for tissue dissociation using the Papain Dissociation System (cat #LK003150, lot #35S16330; Worthington® Biochemical Corporation, New Jersey, USA) following the manufacturer's protocol, with slight modifications; the EBSS solutions and the medium solution with serum were adapted from Saxena et al. (DOI: 10.2144/0000113878). To ensure the exclusive isolation of live nucleated cells, cell suspensions were incubated with 2 μg/ml of Hoechst 33342 (a UV fluorescent adenine-thymine binding dye; #H1399, Life Technologies) at 37°C for 15 min. In order to exclude dead cells, 1 μM of DRAQ7 (a far-red fluorescent DNA intercalating dye; #DR71000, BioStatus) was added to the cell suspensions before fluorescence activated cell sorting (FACS). Approximately 80000 Hoechst+/DRAQ7- cells were collected from each single-cell suspension pool, each in a final volume of 100μl. After FACS sorting, cell suspensions were concentrated at 800 cells/microliter before loading on the 10X Genomics Chromium controller. For a detailed description of the protocol, please refer to the methods section of Flores Horgue et al. (2021). cDNA libraries were constructed following the 10X Genomics Chromium Next GEM Single Cell 3' v3.1 protocol (dual index libraries).