TG isolation and single-cell preparation

There are several approaches to dissecting TG tissues. One of them is to collect the TG with surrounding dura and another is to isolate the dura-free TG. We have isolated TG with surrounding dura for scRNA-seq, TG without dura for IHC, and both preparations of TG with and without dura for flow cytometry. Briefly, prior to TG dissections, the animals were perfused with cold phosphate-buffered solution (PBS) to eliminate the contributions of immune cells from blood to the scRNA-seq and flow cytometry data. For IHC, mice were perfused with 4% paraformaldehyde prior to tissue dissections. TG were dissected from the skull base after the removal of the brain. For dura-free TG, V1–V3 were cut close to TG, and then dura-free TG was lifted by a spatula. For TG with dura, continuous cuts were made all around TG, resulting in a dissected TG covered by dura.

Mice were perfused with PBS to flash out the blood cells from the tissues, including TG. Dissected TG were collected in ice-cold HBSS buffer and subjected to preparation of single-cell suspension for scRNA-seq or flow cytometry. Single-cell suspension was generated using Liberase and Dispase II as described previously (16). After this step, single-cell suspension was processed in two different ways. For the first scRNA-seq experiment, fractions enriched with sensory neurons were obtained using the Percoll gradient as described previously (16). For the second scRNA-seq experiment, viable TG cells were purified by flow cytometry using Calcein Violet-AM/Helix NP NIR (BioLegend) dual live/dead stain. Calcein Violet-AM is a cell-permeable fluorescent probe cleaved and activated in live cells by esterases, whereas Helix NP NIR is impermeable to live cells and detects the nucleic acids of dead cells. Briefly, TG cells were stained first with 0.1 μM of Calcein Violet-AM for 40 min at room temperature, followed by 5 nM of Helix NP NIR. Calcein Violet-AM⁺/Helix NP NIR-gated TG cells were sorted to DMEM/5% fetal calf serum (FCS) medium using the BD FACSAria Fusion cell sorter equipped with a 100 μm nozzle. Sorted cells were centrifuged and resuspended in 15 μl of 1X PBS, 0.04% BSA, and 0.1 U/μl RNase inhibitor. For flow cytometry experiments, single-cell suspensions after the Liberase–Dispase step (see above) were stained with a panel of antibodies as described below.