Supplementary Figure 5: Hox genes are upregulated in Smchd1-null NMPs differentiated from mESCs.

a. Principal component analysis (PCA) of RNAseq libraries from the differentiation of Smchd1^fl/fl and Smchd1^del/del mESCs to NMPs. Samples separate by timepoint on the first component (45%, x-axis) and by coverage on the second (22%, y-axis). Smchd1^fl/fl samples are depicted in shades of red, and Smchd1^del/del samples are depicted in shades of blue. Darker colours represent early time points, whereas lighter colours represent later time points. b. Dot plots showing expression of all genes in Smchd1^fl/fl (x-axis) compared with Smchd1^del/del (y-axis) in log(RPM) at D1, D5, and D8 post-induction of NMP differentiation. All genes are depicted in grey. Differentially expressed genes (FDR < 0.05) are depicted in blue. Hox genes are depicted in red. Hox genes which are differentially expressed are depicted in green. Pearson’s correlation value (R) is shown on the bottom, right of each scatter plot. c. Line graphs showing Hox gene expression throughout in vitro differentiation of male Smchd1^fl/fl and Smchd1^del/del mESCs into NMPs, as shown in Fig. 3a. Each graph shows expression for one paralogue group. i.e. the top left graph is showing expression of the Hox1 gene in three paralogous clusters. Time in days (x axis) is plotted against log₂ transformed reads per million (RPM) (y axis). Smchd1^fl/fl values are plotted in blue, and Smchd1^del/del in red. Circles represent genes from the HoxA cluster, squares represent HoxB, triangles represent HoxC, and upside-down triangle for those in HoxD. Solid lines depict genes that are differentially expressed (FDR < 0.05) at D8 between Smchd1^fl/fl and Smchd1^del/del NMP. Dotted lines indicate genes that were not called as differentially expressed using edgeR. d. Line graphs showing the downregulation of 4/5 pluripotency genes between day 1 and day 8 of the differentiation series in Smchd1^fl/fl and Smchd1^del/del samples.