Single cell isolation and flow cytometry for scRNA-Seq from utricular sensory epithelia

1. Sacrifice E15 chicken embryos by rapid decapitation.

2. Use ice-cold Medium 199 containing Hank’s salts (Gibco) for dissection (M199+). Place the head on a flat surface and bisect the head with a razor blade.

3. Remove the brain, exposing the temporal bones. Use the scissors to remove extra bone and cartilage to isolate the temporal bone form the head.

4. Place the temporal bone in a black Sylgard dish containing ice-cold M199+.

5. The utricle is located under the medial region of the temporal bone. Use forceps to gently open the bony labyrinth to expose membranous labyrinth including the utricle. After opening the temporal bone, identify the utricle by the bright white otoconial mass located on top of the sensory epithelium.

6. Use forceps to detach the utricle from the surrounding membranous labyrinth and nerve tissue. Pull the utricle out and immediately transfer it into a black Sylgard dish containing ice-cold M199.

7. Always transfer utricles with a Moria #35 stainless steel spoon, preventing exposure to air.

8. Use forceps to remove the utricle roof. Remove the otolithic membranes using an eyelash or a gentle fluid stream generated with a 1 ml syringe and attached 30-gauge, 1/2-inch hypodermic needle. Trim away the outside non-sensory epithelium edges using a sapphire knife.

9. Incubate utricles in thermolysin (0.5 mg/ml; Sigma) in M199+ for 20 minutes at 37°C; inactivate in M199+ that contains 10% FBS.

10. If desired, sample cells from either the lateral or the medial side by cutting along the anterior-posterior axis of the utricle using a sapphire knife. The lateral and medial halves should be kept separately subsequently.

11. Carefully peel off the sensory epithelia from the underlying stromal cells using a 30-gauge, 1/2-inch hypodermic needle attached to 1 ml syringe.

12. Transfer the sensory epithelia with a spoon into an 1.5 ml Eppendorf tube containing Accutase (Innovative Cell Technology). Dissociate the sensory epithelia for 20 min at 37°C.

13. Carefully mechanically triturate the dissociating cells using a 1000 µl pipettor, then wash twice with PBS using centrifugation for replacement of buffer (300g, 5 min). Resuspend the cell pellet in 300 µl PBS and keep on ice for flow cytometry.

14. Sort cells with a FACSARIA II instrument (BD Biosciences) set to “single cell” mode and equipped with a 100 µm nozzle.

15. Remove debris based on side-scatter area (SSC-A) and forward-scatter area (FSC-A)

16. Discard doublets using two subsequent gating steps: forward-scatter height (FSC-H) versus forward-scatter area (FSC-A), and side-scatter width (SSC-W) versus side-scatter area (SSC-A).

17. Use SYTOX® Red Dead Cell Stain (Molecular Probes) to identify live and dead cells (SSC-A versus SYTOX Red).

18. Prepare collection wells of 96-well PCR plates (USA Scientific) beforehand with 4 µl lysis solution containing 1 U/µl of recombinant RNase inhibitor (Clontech #2313B), 0.1% Triton X-100 (Thermo #85111), 2.5 mM dNTP (Thermo #10297018), 2.5 µM oligo d(T)30 VN (5’-AAGCAGTGGTATCAACGCAGAGTACT30VN-3’, IDT).

19. Based on the final gating strategy (SSC-A versus FITC-A, the channel use for FM1-43), deposit single cells or single FM1-43_High, FM1-43_Middle, and FM1-43_Low cells into individual wells).

20. Immediately seal, freeze on dry ice, and store at -80°C all plates containing sorted cells.

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