Retrograde labelling and RNA deep sequencing of olfactory bulb projection neurons [bulk RNA-seq]

To characterize the molecular diversity of olfactory bulb projection neurons we used viral targeting and Fluorescence Activated Nuclei Sorting (FANS) to enrich for piriform cortex-projecting or AON-projecting neurons, and bulk RNA deep sequencing (bulk RNA deep seq) to comprehensively characterize their transcriptomes. Overall design: To isolate GFP-labelled nuclei 6 individual replicates for each injection type were used (3 for AON and 3 for PCx-injected mice). 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).