Extended Data Fig. 3. RNA-seq upon m⁶A depletion reveals upregulation of autosomal but not X-chromosomal transcripts.

a. Principal component analysis indicates high reproducibility of RNA-seq data for male mESC in control and m⁶A-depleted conditions (STM2457, 24 h, 4 replicates per condition, total of 398 million uniquely mapped reads). Replicate number given next to each data point. b. Correlation of expression fold changes (log₂) of RNA seq data in m⁶A-depleted (STM2457, 24 h) over control conditions using normalisation to ERCC spike-ins (x-axis) or 100 randomly chosen genes without m⁶A sites (y-axis, see Methods; two-sided Pearson correlation coefficient [R] = 1, P value < 2.2e-16). c. Upregulation upon m⁶A depletion increases with the number of m⁶A sites in the transcripts. Distribution of fold changes (log₂) in m⁶A-depleted (STM2457, 24 h) over control conditions in expressed transcripts (transcripts per million [TPM] > 1, based on total miCLIP2 signal) stratified by their number of m⁶A sites. Numbers of transcripts in each category are indicated above. Boxes represent quartiles, centre lines denote medians, and whiskers extend to most extreme values within 1.5× interquartile range. d. Cumulative distribution of expressed autosomal (grey) and X-chromosomal (orange) transcripts (RPKM > 1) with a given expression level (RPKM, x-axis). The expression distributions of X-chromosomal and autosomal transcripts are largely identical, supporting a X:A ratio close to 1 across the full expression range. For comparison, a theoretical doubling of the X-chromosomal expression is shown (orange, dotted) which would exceed autosomal expression levels. e. Median X-to-autosome (X:A) expression ratios increase with higher RPKM cut-offs (>0, n [genes] = 26,291, ≥0.25, n = 13,795, ≥0.5, n = 12,255, ≥1, n = 10,849). Median X:A ratios for male mESC and 95% confidence intervals are given.

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