a, Generation of aneuploid cells through random meiotic segregation from a homozygous triploid (3N) yeast strain that bears a centromeric plasmid that carries the selection marker spHIS5 under the MATa-specific promoter STE2pr. Coloured rods represent chromosomes. b, Left, DNA content of a cohort of random aneuploid spore colonies (each represented by a grey dot, n = 75; see Source Data) produced through triploid meiosis and analysed by FACS; box plot and violin plot (defined as in Fig. 2 and Supplementary Methods) show the distribution of ploidy levels. Right, 192 random aneuploid colonies produced by triploid meiosis were pooled, and the resulting population subjected to quantitative PCR-based karyotyping (representative results from two biological repeats). Y axis represents the relative copy number (mean of the two arms) of each chromosome (dots) to the reference haploid yeast. There was no significant difference in copy number between each chromosome (one-way analysis of variance, P value = 0.998). c, Principal component analysis of RNA sequencing (RNA-seq) results in different populations. PC1 (x axis) shows the apparent difference between haploid (n = 2) and aneuploid (n = 5) populations. PC2 (y axis) reflects a small difference in the two methods of generation of aneuploid populations. Each dot represents one population. d, Gene expression in heterogeneous aneuploid populations (n = 5) relative to haploid populations (n = 2) in MA plot. x axis represents basal mean expression in log 10 scale; y axis represents differential expression changes between aneuploid and euploid populations in log 2 scale. P values calculated on the Wald statistic were corrected for multiple comparisons (Benjamini–Hochberg), and the resulting false discovery rate was further corrected for variance underestimation using an empirical null model. CAGE genes were identified as genes with final false discovery rate < 0.05 (see details in Supplementary Methods; exact P values in Supplementary Table 1). Each dot represents 1 gene, and 222 significantly differentially expressed (CAGE) genes common to all 5 aneuploid cell populations are labelled in dark red. e, Quantitative PCR with reverse transcription (RT–qPCR) validations of four significantly differentially expressed genes from the RNA-seq analysis in a heterogeneous aneuploid population (n = 1), and randomly picked individual aneuploid clones (n = 12). Black circles, individual aneuploid populations; red dot: a heterogeneous aneuploid population; green dot, aneuploid populations in RNA-seq (d). For individual aneuploid colonies, the expression change of each gene was normalized by the gene copy number (determined by qPCR), and then normalized to that of a non-CAGE gene (ACT1). Average gene-expression changes in the population and in the individual aneuploid clones followed similar trends (up- or downregulated) to those of heterogeneous populations in RNA-seq. Box plot representation as in Fig. 2 for measurements from the 12 aneuploid clones. f, Correlation analysis of expression changes of the genes implicated in the CAGE signature, showing positive correlation between aneuploid populations and stable aneuploid strains5 (n = 5) (Spearman’s rank correlation). Green circles represent mean expression changes of genes within the CAGE signature (213 in common in the transcriptomic data between aneuploid populations and the individual strains). Error bars, s.e.m. Only eight genes across all five strains appear to show opposite expression trend to CAGE. The trend line was fitted by linear regression (red dashed line) with grey ribbon (95% CI). Significant positive correlation was observed between the average gene-expression changes in the five aneuploid strains, and CAGE observed with the heterogeneous aneuploid population subjected to RNA-seq (P value = 1.609 × 10−7). Source Data