Validation of molecularly-defined olfactory bulb projection neurons targeting anterior or posterior brain areas

To validate different projection targets of already molecularly-defined olfactory bulb projection neurons we used viral targeting specifically into anterior or posterior cortical areas, Fluorescence Activated Nuclei Sorting (FANS) to enrich for olfactory bulb projection neurons, and single-nuclei RNA sequencing (sn-RNA seq) To isolate GFP-labelled nuclei, 1 individual replicate of AON or PCx-injected mice was used. Ipsilateral and controlateral sides were minced separately and placed into two different tubes. The minced tissue was gently homogenized in Nuclei PURE Lysis Buffer and 10% Triton X-100 using an ice-cold dounce and pestle, and filtered two times through a 40 µm cell strainer on ice. After centrifuging at 500 rpm for 5 min at 4 °C, the supernatant was aspirated and gently resuspended in 500 µl of cold buffer (1x of cold Hanks' Balanced Salt Solution HBSS, 1% nuclease-free BSA, RNasin Plus and 1/2000 DRAQ5). Our study identifies molecularly distinct subtypes of mitral cells projecting to anterior or posterior olfactory cortices. Overall design: we used AAV viral targeting and Fluorescence Activated Nuclei Sorting (FANS) to enrich for olfactory bulb projection neurons, and single-nuclei RNA sequencing (10x Genomics)