GSM7989134: S80_E14_vestB; Mus musculus; RNA-Seq
Utricles from 5 mice were microdissected in ice-cold DMEM/F12 media and collected in separate tubes, on ice. The pooled tissue was then placed in a pre-warmed dissociation buffer of EBSS solution containing 33 units of papain (Worthington Biochemical), 1.67 mM L-cysteine, 0.83 mM EDTA, 15 mM HEPES, and 10 Kunitz/ml DNase I. The tissues were incubated for 45 – 60 min at 37°C, triturating with a 200 μL pipette every 10 min for tissue dissociation.  At the end of the 37°C incubation, the solution was passed through a 25G needle a few times to ensure a single cell suspension. An equal volume of DMEM/F12 media containing 20% ovomucoid protease inhibitor (Worthington Biochemical) was added, and the dissociated cells were passed through a 40 μm filter (pluriSelect) to remove debris. The cells were pelleted at 300 g for 5 min, washed twice with DMEM/F12 containing 0.04% BSA, and then pelleted and resuspended in the DMEM/F12 with BSA solution. The cells were counted with the Luna-FL automated fluorescence cell counter using an AO/PI viability assay (Logos Biosystems) and immediately loaded on the 10x Chromium controller (10x Genomics) for partitioning single cells into droplets. For each sample, 15,000 cells were loaded in one or two channel(S) of a Chromium Chip (10x Genomics). 3' v3 chemistry was used to profile all other samples. cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). cDNA and gene expression library fragment sizes were assessed with a DNA High Sensitivity Bioanalyzer Chip (Agilent). cDNA and gene expression libraries were quantified using the Qubit dsDNA High Sensitivity assay kit (Thermo Fisher Scientific). Gene expression libraries were multiplexed and sequenced on the Novaseq 6000 (Illumina) with the following read structure: Read 1: 28 cycles, Read 2: 72 - 100 cycles, Index Read 1: 8 cycles.