GSM6594423: Vestibular adult; Homo sapiens; RNA-Seq
Human fetal inner ear tissue was stored in RNAlater (AM7020, Invitrogen) at 4°C and processed to a single nuclei suspension within 16 h after collection. Human adult tissue was processed within 2 h after collection. After collection of the inner ear tissue, the tissue was evaluated using a dissection microscope (M205C, Leica). The otic capsule of the W9.2 fetal inner ear was removed. The tissue was washed once with ice-cold PBS, and resuspended in lysis buffer that contained 10 mM Tris-HCl (T2194, Millipore-Sigma), 10 mM NaCl (59222C, Millipore-Sigma), 3 mM MgCl2 (M1028, Millipore-Sigma), 0.005% Nonidet P40 Substitute (74385, Millipore-Sigma) in DEPC-treated water (750024, Invitrogen). The suspension was transferred to a Dounce tissue grinder (885300-0002, Kimble) and ground every 5 min. After 20 min, dissociation to single nuclei was confirmed by bright-field visualization (EVOS M5000, Thermo Scientific). After trituration using p1000 and p200 tips, the suspension was filtered through a 70 μm MACS SmartStrainer (130-098-462, Miltenyi Biotec), centrifuged at 500xg for 5 min at 4°C and resuspended in 1% BSA in PBS (AM2616, Invitrogen) containing 200 U/μL RNase inhibitor (3335402001, Millipore-Sigma). After filtration using a 40 μm Flowmi cell strainer, as well as filtratration through a 20 μm and 10 μm Pluristrainer (43-10020-40 and 43-10010-40, PluriSelect), the nuclei suspension was resuspended to a final concentration of 1,000 cells/μL. Single nucleus gene expression libraries were generated on the 10x Genomics Chromium platform using the Chromium Next GEM Single Cell 3' Library & Gel Bead Kit v3.1 and Chromium Next GEM Chip G Single Cell Kit (10x Genomics) according to the manufacturer's protocol.  Gene expression was sequenced on a NovaSeq 6000 S4 flow cell using v1 chemistry (Illumina) and FASTQ files were generated using Cell Ranger mkfastq (10x Genomics).