Extended Data Fig. 1: An INTACT-FACS-snRNA-seq system for high throughput transcriptomic analysis of cell types in adult mouse cerebellum.

a, Adult Pcp2-Cre/Sun1^fl/wt mouse cerebellum was subjected to immunohistochemical analyses with antibodies labeling Calbindin (red), GFP (green) and the DNA dye bisbenzimide (Hoechst). INTACT labeling was restricted to Calbindin+ Purkinje neurons. Scale bar, 100 μm. Similar results were found in 4 animals for representative data. b, Schematic representation of the sagittal view of lobules across the central vermis and top view of the whole cerebellum. Parasagittal Zebrin II+ strips are labeled in light color and anatomically dissected anterior vermis lobules are boxed. c, Adult mouse cerebellum was subjected to INTACT gradient nuclei isolation followed by FACS purification. GFP positive or negative while DyeCycle Ruby labeled single nuclei were sorted and defined as qualified for future sequencing analysis. d, Nuclei from pre- and post-FACS were subjected to DNA dye bisbenzimide analysis under the confocal microscope. Preserved intact nuclei after FACS were observed. Scale bar, 10 μm. Similar results were found in 2 animals for representative data. e, f, Nuclei from pre- and post-FACS were subjected to qRT-PCR analyses, and the mRNA expressions of Pcp2 and Gabra6 were either enriched or de-enriched in post-FACS nuclei samples. Data are presented as mean ± s.e.m. ***p = 6.9e-6, 5.6e-6, by two-tailed unpaired t-test, n = 4 mice. g, h, i, Pre- and post-FACS nuclei from cerebellum of baseline mice or mice with rotarod activation were subjected to qRT-PCR analysis of the Fos, FosB and Npas4 expressions. Data are presented as mean ± s.e.m. ***p = 1.9e-5, 2.7e-7 for pre- and post-FACS Fos expression; ***p = 3.0e-4, 3.7e-4 for pre- and post-FACS FosB expression, **p = 0.003, 0.003 for pre- and post-FACS Npas4 expression, n = 4 mice, two-tailed unpaired t-test. j, 18,151 nuclei from anterior vermis lobules assigned into granule neurons, Purkinje neurons, interneurons, glial cells and oligodendrocytes based on unsupervised clustering and UMAP plot visualization. k, l, Nuclei of two biological replicates and sequencing batches from the whole cerebellum or anterior vermis lobules showed similar cell type clustering and high correlation of gene expression. m, Feature plot demonstrated the number of nCounts in each categorized nuclei isolated from the whole cerebellum or anterior vermis lobules. n, Feature plot demonstrated that the GFP transcripts almost exclusively enriched in Pcp2 featured Purkinje neurons. 98.1% GFP+ cells in the whole cerebellum and 95.9% in the anterior vermis lobules were clustered with the Purkinje neuron population. o, Heat map showing identified marker genes in each categorized cell types.