GSM5771272: LFHT1_ROW25_17; Mus musculus; RNA-Seq
3' end single-cell RNA sequencing using Fluidigm's C1 HT IFC with 5bp UMIs and 800 capture chambers OMP(GFP/wt) males aged of 66 days were used (n=7). Mice were anesthetized using isoflurane and euthanized by decapitation. VNOs were dissected and their cartilaginous envelope were discarded. The tissues were immediately placed in ice-cold oxygenated artificial cerebro-spinal fluid (ACSF) with an osmolarity of 300 mOsm and pH at 7.4. After microdissection, the tissues extracted from the 7 mice were pooled into two tubes (i.e. 3 and 4 mice, respectively). Tissue dissociations were performed 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 6000 GFP+/Hoechst+/DRAQ7- cells were collected from each single-cell suspension pool, each in a final volume of 15 μl. After FACS sorting, 4 μl of C1 Suspension Reagent (Fluidigm) was added to the 10 μl of cell suspensions, yielding mixes of 300 cells/μ