GSM6556051: Gallus gallus utricle, cell-159; Gallus gallus; RNA-Seq
Utricles were dissected in ice-cold Medium 199, otolithic membranes were mechanically removed, and tissue was incubated in thermolysin (0.5 mg/mL M199 medium) for 20 minutes at 37°C. Thermolysin activity was attenuated with 10% FBS in M199. The sensory epithelia were carefully peeled off from the underlying stromal cells using a 30- gauge 1⁄2-inch hypodermic needle attached to a 1 mL syringe. For each experiment, we pooled 4-8 sensory epithelia. The epithelia were dissociated using 1X Accutase for 20 minutes at 37°C, followed by mild mechanical trituration, and washed twice with PBS using a centrifugation step (300 x g, 5 min, room temperature (RT)). Viable single cells were isolated with a Becton Dickinson FACSAria Fusion flow cytometer (Ellwanger et al., 2018). Two independent batches of 270 cells were deposited into individual wells of 96-well plates, prefilled with 4 μl of a premade lysis solution with 1 U/μl of recombinant RNase inhibitor, 0.1% Triton X-100, 2.5 mM dNTP mix, 2.5 μM oligo d(T)30 VN (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3', IDT). Plates containing sorted cells were immediately sealed, frozen on dry ice, and stored at −80°C. Single-cell RNA-seq was performed via Picelli and colleagues' method (Picelli et al., 2014) using SMARTscribe for reverse transcription followed by 22 amplification cycles. Amplified cDNAs were purified by AMPure Beads cleanup using a Biomek FX automated platform and assessed with a fragment analyzer (Agilent) for quantitation and quality assurance. Barcoded libraries were synthesized using a scaled-down Nextera XT protocol (Mora-Castilla et al., 2016) in a total volume of 4 μl. A total of 384 libraries were pooled, and paired-end sequenced (2 x 150 bp) on a NextSeq 500/550 High Output flow cell.