Figure 1. Overview of the study and changes in total cellular RNA content.

(a) Microphotographs and number of cells used from each developmental stage studied. A total of 348 cells were collected at oocyte and zygote stage, day 2 (4–6 blastomeres) and day 3 (7–10 blastomeres) after fertilization. Scale bar, 20 μm. (b) Heterogeneity of gene expression among oocytes, day 2 and day 3 embryo blastomeres. The heatmap shows Spearman correlation coefficient (in complete pairs of observations) between the samples in library L185. The correlation matrix is sorted by hierarchical clustering with Euclidean distance and complete linkage method. (c) Spearman correlation coefficient between the samples in library L186 showing biological variation in day 2 and day 3 blastomeres. (d) Comparison of relative poly(A)-tailed RNA content between the developmental stages from plates L233, L185 including exactly staged 4-cell embryos (n=23), and L186 including exactly staged 4-cell (n=11) and 8-cell (n=22) embryos. Relative poly(A)-tailed RNA content per cell is calculated as the ratio of total mapped reads on genome references to mapped reads on spike-in references with centering by average of the ratios in earlier stage. Red line indicates average level, and P value is the assessment of the mean rank difference by Wilcoxon rank sum test. (e) A comparison of overall proportion of TFEs according to genome annotation. The comparisons are made between oocyte to zygote, oocyte to 4-cell, and 4- to 8-cell. The annotated groups marked with dots or crosses and separated by black lines are plotted separately in f. (f) Proportion of annotated reads for functional transcripts from groups ‘upstream and 5′UTR of coding', ‘upstream and 1st exon of noncoding', and ‘intron' and ‘unannotated' in the transitions from oocyte to 4-cell, 4- to 8-cell and comparison of 8-cell stage blastomeres to human brain RNA. Purple lines indicate average ratio, and P value is the assessment of the mean rank difference by Wilcoxon rank sum test. The variation between 8-cell stage blastomeres was more than pure technical variation represented by human brain samples, suggesting biological variation. 4-cell, four-cell; 8-cell, eight-cell.