Extended Data Fig. 5: AraC generates DSBs in Golgi and Purkinje cells in the cerebellum.
a, Flow cytometric analysis of nuclei isolated from 1 month old mouse cerebellum after PBS- or AraC treatment. Y- and X-axes indicate the expression of the Purkinje specific marker ITPR1 and the DNA damage marker γ-H2AX respectively. Purkinje cells and an unknown population are marked by the red and green box respectively. For the flow cytometry gating strategy, see Supplementary Fig. 2. b, Cell type composition of sorted Purkinje cells and the unknown population shown in (a) determined by RNA-seq. The estimated fraction is determined using single-cell sequencing data from mouse brain. Nuclei were isolated 3 h after AraC treatment. c,d, Top 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified from AraC-induced upregulated (c) and downregulated (d) genes in Purkinje cells. Statistical significance was determined using the one-sided hypergeometric test. e, Fos expression in Purkinje cells isolated from wild type mice receiving PBS or AraC. Statistical significance was determined using the two-sided Wald test and adjusted by the Benjamini & Hochberg method, p value = 0.03. Data are presented as mean ± SEM, six and five mice were used for analysis in the PBS and AraC groups, respectively. f, Flow cytometric analysis of nuclei isolated from the whole brain without cerebellum from PBS- or AraC-treated mice. Y- and X-axes represent the expression of the DNA damage marker γ-H2AX and the neuronal marker NeuN respectively. The NeuN and γ-H2AX positive population is highlighted in the red square. For the flow cytometry gating strategy, see Supplementary Fig. 2. g, Cell type composition of sorted γ-H2AX positive population upon AraC treatment shown in (f) determined by RNA-seq. The estimated fraction is determined using single-cell sequencing data from mouse brain. h, Bar graph depicting the percentage of (5fC + 5caC)/total C in 55 CpGs at seven sites in genes Grid2 (six sites) and Itpr1 (one site) in Purkinje nuclei isolated from wild type or Pcp2-Cre TDGfl/fl mice (Tdg−/−) as measured by PS-seq. i, Integrative Genomics Viewer (IGV) Screenshots of H3K27ac and RNA-seq for highly expressed genes in Purkinje cells (Itpr1 and GABBR2), Golgi cells (SGCD and TCF4), and highly expressed genes in both Purkinje and Golgi cells (KCNMA1 and FRMD4A). j, Aggregate END-seq signal at DNA methylation valleys (DMV) within 118 AraC-induced downregulated genes.
