GSM9019650: Larynx, TdTomato Red sorted cells isolated from 10 murine larynges, rep 1 (5x males and 5x females); Mus musculus; RNA-Seq
Mouse larynges were collected from Lrig1CreERT2/+; ROSA26CAG−LSL−tdTomato/+ , WT, and ROSA26CAG−LSL−tdTomato/+ mice. Tissues were minced into 0.5 mm pieces under a dissection scope and dissociated using the protocol by An et al. Dissociation buffer contained Accumax, Bacillus lichenformis protease, collagenase I, and DNase I in Ca/Mg-free PBS. Each larynx was digested in 300 μL solution at room temperature for 5 min, followed by 15 min vortexing on ice with gentle trituration. Cells were pooled, centrifuged, resuspended in DPBS with 10% FBS, and filtered through 40 μm strainers. ACK lysis buffer was used to remove erythrocytes. For FACS, cells were washed, filtered again, and sorted using a FACSAria2 (BD Biosciences). WT and background controls were used to set gates. Sorted cells were analyzed using J Flow software. Library construction was performed following the manufacturer's protocol using the Chromium GEM-X Single Cell 3' v4 Reagent Kit (10x Genomics). Cells in single-cell suspensions were delivered on ice to the sequencing center, where cell concentration and viability were measured with the Luna-FX7 (Logos Biosystems) using acridine orange/propidium iodide staining. Single cells were encapsulated in GEMs, and reverse transcription and barcoding were carried out using an Eppendorf MasterCycler Pro thermocycler. Amplified cDNA was purified using SPRIselect beads (Beckman Coulter), quantified using a Bioanalyzer 2100 (Agilent Technologies) with the High Sensitivity DNA kit, and used for library preparation. Sequencing was performed on the Illumina NovaSeq X Plus system with 150 bp paired-end reads.