GSM5124293: Mouse cochlea neurons, naive 3; Mus musculus; RNA-Seq
Neuronal tissue: Temporal bones from naïve (n = 4) and noise exposed (n = 4) 9 wko, male CBA/CaJ mice were isolated in ice-cold Leibovitz's L-15 medium. The overlying bone and lateral wall were extracted from apex to base, leaving just the modiolus, with the spiral ganglion, the spiral limbus, inner sulcus, and portions of the organ of Corti and outer sulcus remaining. Microdissected tissue from each mouse was pooled in a solution of Leibovitz containing 200 units/mL collagenase IV (Sigma) and 10 kunitz/ml DNase I (Stem Cell Technologies) and incubated for 30 min at 37 °C to digest extracellular matrix. The collagenase IV solution was replaced with EBSS containing 20 units of papain (Worthington Biochemical), 1 mM L-cysteine, 0.5 mM EDTA, 15 mM HEPES, and 10 kunitz/ml DNase I and incubated for an additional 30 min at 37 °C, triturating with a 1000 µL pipette at 10 min intervals to generate a single cell suspension. An equal volume of Leibovitz containing 20% ovomucoid protease inhibitor (Worthington Biochemical) was added, and the cell suspension passed through a 20 m filter (pluriSelect) to remove debris. The cells were pelleted at 300g for 5 min, washed twice with PBS containing 0.04% BSA, then pelleted and resuspended in PBS/BSA solution. Finally, a 9 µL sample of the cell suspension was counted on a Luna Fl automated counter using an AO/PI fluorescent cell viability assay (Logos Biosystems).   Lateral wall tissue: Temporal bones from naïve (n = 4) and noise exposed (n = 4) 7-8 wko, male CBA/CaJ mice were isolated in ice-cold Leibovitz's L-15 medium. Using a #11 scalpel, the bony wall of the cochlea was removed to reveal the cochlear lateral wall.  The lateral wall with attached stria vascularis was extracted from apex to base and separated from the modiolus and other cochlear tissue. Microdissected tissue was then dissociated into single cells using the collagenase IV and papain enzyme mixtures as described for the dissociation of SGN tissue.  For the strial tissues, however, the total incubation time in papain was extended to 1 hr to ensure complete tissue dissociation. After cell counting, the appropriate volume of cell suspension was added to target capture of 10,000 cells per sample using the high throughput, droplet microfluidics Gemcode platform from 10X Genomics with v2 chemistry (Zheng et al., 2017). Each droplet on the Gemcode co-encapsulates a cell and a gel bead that is hybridized with oligo(dT) primers encoding a unique cell barcode and unique molecular identifiers (UMIs) in lysis buffer. The capture process takes 6 min, after which the transcriptomes captured on gel beads are pooled and reverse transcribed to cDNA. RT, PCR amplification of cDNA, and preparation of a library from 3' ends were conducted according to the manufacturer's published protocol. We performed 15 cycles of PCR amplification of cDNA. The libraries were sequenced on an Illumina NovaSeq 6000 at the Broad Institute Sequencing Facility.