Extended Data Fig. 3: High-throughput isolation of retrogradely labelled spinal projecting neurons for snRNA-seq.

a, Regions containing retrogradely labelled SPNs were dissected from P56 mice. A 1 mm coronal-oriented brain matrix was used to section brains from the olfactory bulb to Lambda, and a sagittal-oriented matrix was used to section the remaining subcortex. Test tubes were drawn using templates from Servier Medical Art (Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licences/by/3.0/). Sagittal atlas outline adapted from Allen Institute for Brain Science (https://mouse.brain-map.org/static/atlas)⁵³. b, Dissection scheme was based on STPT imaging, which shows the cleanest dissection planes to separate the major regions containing SPNs are coronal for forebrain (i) and sagittal for midbrain and hindbrain (same as panel Fig. 1b) (ii). Equal numbers of male and female mice were pooled per ROI to input sufficient nuclei for FANS (iii). Panel b is same as in Fig. 1b. c, Example dissecting microscope images showing the dissection planes based on the scheme in a and b. Estimated scale based on Paxinos adult mouse brain atlas, ~1000 µm. This scheme was used to dissect the N = 30 mice from which the snRNA-seq datasets were generated (donor information summarised in Supplementary Table 1). d, Following microdissection, nuclei were isolated from each ROI and SPNs enriched using FANS. Representative plots shown are from an M1M2S1 sample. e, Enrichment for GFP+ and/or mScarlet+ nuclei was confirmed post-FANS. Scale, 100 µm. Box plot depicts the proportion of sorted nuclei (labelled with DAPI) that had detectable GFP and/or mScarlet via widefield microscopy (N = 4 M1M2S1 samples). The centre line of the box plot depicts the median value (50^th percentile) while the box contains the 25^th to 75^th percentiles; whiskers correspond to the 5^th and 95^th percentiles. Rep, replicate.

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