title: GSM8970753: single nucleus RNA sequencing of mouse nodose ganglia gene expression library; Mus musculus; RNA-Seq
library_construction_protocol: For snRNAseq: Mice were euthanized and the nodose ganglia were immediately extracted and snap frozen on dry ice pooling overnight-fasted left, overnight-fasted right, ad libitum fed left, and ad libitum fed right nodose ganglia into four separate tubes. Each sample was separately homogenized in 500µL homogenization buffer in a dounce homogenizer on ice. Samples were centrifuged at 900 r.c.f at 4°C for 10 minutes. The supernatant was removed and pellets were resuspended in a 1:1 mixture of homogenization buffer and 50% Optiprep diluted in iodixanol dilution media. These suspensions were then layered on top of 450µL 29% Optiprep diluted in iodixanol dilution medium and were centrifuged at 13,500 r.c.f, at 4°C for 20 minutes. The supernatant was removed, and pellets were resuspended in 1mL PBS containing 0.04% BSA. Resuspended samples were centrifuged at 900 r.c.f for 10 minutes at 4°C and supernatant was removed. Samples were resuspended in 100µL of different cell multiplexing oligo (CMO; from the 10X Genomics 3' CellPlex kit) and incubated for 5 minutes at room temperature. After incubation, 1.9mL resuspension buffer (PBS with 1% BSA and 0.4U/µL RNasin) was added to each sample, and the mixture centrifuged at 900 r.c.f for 10 minutes, and then the supernatant was removed. Another 1mL resuspension buffer was added, the mixture centrifuged at 900 r.c.f for 4 minutes and then the supernatant removed. Nuclei pellets were resuspended in 100µL resuspension buffer each and then combined. The pooled sample was centrifuged at 900 r.c.f for 10 minutes, the top 50µL of supernatant removed, and the pellet resuspended in the rest of the supernatant. For spatial transcriptomics: The left and right nodose ganglia from six mice were dissected and snap frozen on dry ice. Three nodose ganglia in the same orientation were placed on a single disposable base mould and embedded by adding chilled bubble free optimal cutting temperature compound. The samples, comprising 6 left nodose ganglia and 3 right nodose ganglia in total, were stored at -80°C until sectioned on a cryostat at -20°C into 10µm sections. The 10µm section was mounted to a pre chilled 3mm by 3mm Curio Seeker tile slide for high-resolution spatial transcriptomics. For snRNAseq: libraries were generated by following the Chromium Next GEM Single Cell 3' v3.1 Dual Index protocols, with feature barcode technology for cell multiplexing. For spatial transcriptomics libraries were generated according to the Curio Seeker Spatial Mapping Kit (3x3) user guides.