Extended Data Fig. 4: Characterization of gene expression and m⁶A methylation in the cortex, hippocampus, and cerebellum.

a, Hierarchical clustering of gene expression similarity scores across samples from each brain region expressing either APOBEC1-YTH or APOBEC1-YTH^mut. Samples primarily cluster by brain region, not by which transgene is expressed. b, Volcano plots indicating the number of differentially expressed genes between APOBEC1-YTH-expressing brain regions. Significance was determined using DESeq2 (negative binomal regression followed by Wald test and FDR-based p-value adjustment). c, Volcano plots indicating the small number of gene expression differences between APOBEC1-YTH and APOBEC1-YTH^mut-expressing cells in each brain region. Numbers indicate the number of up/downregulated genes. Significance was determined using DESeq2 (negative binomal regression followed by Wald test and FDR-based p-value adjustment). d, Number of differentially expressed RNAs between the cortex of DART mice and wild type mice^29,62. There are similar numbers of differentially expressed RNAs across all samples, indicating that APOBEC1-YTH expression does not substantially alter gene expression patterns. e, Distribution of %C2U values at m⁶A sites identified in the cortex (n = 17,878 sites from 4 biological replicates), hippocampus (n = 13,461 sites from 4 biological replicates), and cerebellum (11,391 sites from 4 biological replicates). Significance was determined using a two-sided Wilcoxon rank-sum test. Center line in box represents median, while the box represents the 25^th-75^th percentile. Whiskers represent the highest and lowest values (1.5x interquartile range), with outliers shown as dots. Cortex vs. Hippocampus p-value = 0.004. Cortex vs. Cerebellum p-value = < 2.2e-16. Hippocampus vs. Cerebellum p-value = < 2.2e-16. * = p < 0.05. **** = p < 0.0001. f, Metagene analysis showing the distribution of m⁶A sites identified in DART mice in each brain region. Significance was determined using a two-sided t-test with no p-value adjustment. g, m⁶A sites identified in the cortex of DART mice are enriched in long internal exons. Barplot indicates the number of methylated internal exons relative to the total number of internal exons within each group. Solid line at 1 indicates no enrichment. h, Overlap of methylated RNAs identified by in vivo DART-seq and antibody-based m⁶A profiling in each brain region. i, Independent validation of methylation at randomly selected m⁶A sites using RT-qPCR-based relative m⁶A quantification. Dotted line at 1 indicates no detectable methylation. n = 3 biological replicates. Error bars represent standard error. Significance was determined using a one-sided t-test comparing to 1. Mean for Paqr8 = 49.41, mean for Ube2v1 = 6.31, mean for Nova2 = 1,375. j, Overlap of individual m⁶A sites identified across all three brain regions. k, Comparison of APOBEC1-YTH transgene RNA levels across brain regions. The number of normalized counts mapping to rat APOBEC1 is shown. Significance was determined using DESeq2 (negative binomal regression followed by Wald test and FDR-based p-value adjustment). Error bars represent standard error (n = 4 biological replicates). Mean for APOBEC1-YTH samples: Cortex = 2,218; Hippocampus = 2,644; Cerebellum = 1,278. Mean for APOBEC1-YTH samples: Cortex = 2,680; Hippocampus = 2,855; Cerebellum = 1,304. * = p < 0.05. **** = p < 0.0001. l, Bar chart showing the number of overlapping methylated RNAs between DART-seq in the cortex and MeRIP-seq datasets (Liu et al 2020) across mouse tissues. m, Clustering of m⁶A methylation patterns from mouse brain regions and other organs using the DART-seq and Liu et al 2020 datasets. n, Gene expression analysis of m⁶A writers, readers, erasers, and core EJC components derived from RNA-seq data from Liu et al 2020. Log₂FC represents brain relative to other tissues. o, Validation of differential m⁶A levels using RT-qPCR-based m⁶A quantification. Two sites identified with in vivo DART-seq as being differentially methylated between the cortex and cerebellum (n = 4 biological replicates) and two sites differentially methylated between the cortex and hippocampus (n = 2 biological replicates) are validated. Data are plotted as the relative methylation level detected in cortex, normalized to either cerebellum or hippocampus from the same animal. Individual data points are shown with lines indicating brain regions from the same animal. Error bars represent standard error. Significance was determined using a one-sided t-test comparing values to 1. *** = p < 0.01.

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