GSM5770996: LFHT1_ROW12_1; 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/μl. Cells were captured using the C1 Single-Cell mRNA Seq high-throughput (HT) integrated fluidic circuit (IFC) designed for 10-17 μm cells (cat #100-5760, Fluidigm). Each 14 μl mix was loaded on one side of the chip which was processed on the C1 System (cat #100-7000, Fluidigm) following the manufacturer's protocol. Cell lysis, barcoding, reverse transcription and PCR amplification were performed directly on the C1 chip using Fluidigm's C1 Single-Cell mRNA Seq HT Reagent Kit v2 (cat #101-3473, Fluidigm) following the manufacturer's protocol. For a detailed description of the protocol, please refer to the methods section of Fodoulian et al. (2020) bioRxiv (https://doi.org/10.1101/2020.10.14.339259). The cDNA content of each column on the chip (formed of 40 cells) was pooled before harvesting. Twenty cDNA libraries were generated using the Nextera XT DNA Library Preparation Kit (cat #FC-131-1024, Illumina) and Nextera XT Index Kit (cat #FC-131-1002, Illumina) following Fluidigm's protocol. Before the final purification step, all 20 cDNA libraries were pooled. The library pool molarity and quality were assessed with the Qubit 2.0 using the Qubit dsDNA HS Assay Kit (cat #Q32854; Thermo Fisher Scientific) and the TapeStation using the Agilent High Sensitivity DNA Chip (cat #5067-5584; Agilent Technologies). The pool was then loaded at 7 pM on 4 lanes for clustering on a rapid paired-end Illumina Flow Cell (cat #PE-402-4002; Illumina) and sequenced on an Illumina HiSeq 2500 sequencer using the HiSeq Rapid SBS Kit v2 (cat #FC-402-4021; Illumina) chemistry. Read 1 consisted of 11 bases (6 bases for the cell barcode, and 5 bases for the unique molecular identifier), while read 2 was formed of 80 bases.