title: GSM8614765: KO, replicate 2, scRNA seq; Mus musculus; RNA-Seq
library_construction_protocol: P21 male mice (3 Tmie-/-, 4 Tmie+/- controls) were anesthetized, sacrificed by decapitation, and temporal bones were isolated and dissected in ice-cold Leibovitz's L-15 medium. Dissected tissue consisted of the spiral ganglion, spiral limbus, inner sulcus, organ of Corti, and outer sulcus and was pooled in tubes containing ice-cold L-15. Pooled tissue was centrifuged and the supernatant was replaced with a prewarmed solution of Leibovitz containing 200 units/mL of collagenase IV (ThermoFisher, 17104-019) and incubated for 30 min at 37°C. Collagenase solution was replaced with a prewarmed solution of Earle's Balanced Salt Solution (EBSS) containing 20 units/mL papain (Worthington, LK003150), 1mM L-cysteine, 0.5mM EDTA, 15 mM HEPES, 10units/mL DNAse I (StemCell Technologies, 7900), and 8 µL/mL RNAse inhibitor (Lucigen, 30281-1). Single cell solutions were obtained by incubation for 30 min at 37°C and gentle trituration every 10 min via pipette. Then, an equal volume of L-15 medium containing 10 mg/mL ovomucoid inhibitor, 10 mg/mL BSA (Worthington), and 8 µL/mL RNAse inihibtor was added, and the suspension was passed through a 20 µm filter to remove debris. Cells were re-pelleted at 350g for 5 min, washed twice with DMEM/F12 containing 0.04% BSA and 8 µL/mL RNase inhibitor, and pelleted again. Lastly, cells were resuspended in 60 µL of DMEM/F12 containing 0.04% BSA and 8 µL/mL RNase inhibitor. Cell counts and viability were determined using the Cell Countess II with Trypan Blue. Cells were combined with RT reagents and loaded onto Next GEM Chip G along with 3' v3.1 gel beads (10X Genomics, PN-1000121). The NextGEM protocol was run on the 10X Chromium Controller to create Gel Beads in emulsion (GEMs), composed of a single cell, uniquely barcoded gel bead, and reverse transcriptase reagents. Next, 100 µL of emulsion was retrieved from the chip and incubated (45 min at 53°C , 5 min at 85°C, cool to 4°C), generating barcoded cDNA from each cell. The GEMs were broken using Recovery Agent and cDNA was cleaned, following manufacturer's instructions using MyOne SILANE beads (Invitrogen, 37002D). cDNA was amplified for 11-12 cycles based on input cell number (3 min at 98°C, 11-12 cycle:  15 sec at 98°C, 20 sec at 63°C, 1 min at 72°C; 1 min at 72°C, cool to 4°C) and samples were cleaned using 0.6X SPRIselect beads (Beckman Coulter, B23317). QC was completed using Qubit and Bioanalyzer to determine size and concentrations. 10 µL of amplified cDNA (containing 172-222 ng cDNA) was carried into library prep. Fragmentation, end repair and A-tailing were completed, and samples were cleaned up using double sided size selection (0.6X, 0.8X) with SPRIselect beads.  Adaptor ligation (15 min at 20°C, cool to 4°C), 0.8X cleanup and amplification were performed, with PCR using unique i7 index sequences.  Libraries underwent a final cleanup using double sided size selection (0.6X, 0.8X) with SPRIselect beads.  Library QC was performed using Qubit, Bioanalyzer and KAPA library quantification qPCR kit.  Libraries were sequenced on the geSa using v1.5 kits, targeting 50K reads/cell, at read lengths of 28 (R1), 8 (i7), 91 (R2).
identifiers.namespace: GEO
identifiers.id: GSM8614765_r1