Figure 5

Changes in nascent transcription upon Spt5 depletion correlate with mRNA abundance

A. Volcano plot of mRNAseq data, performed in triplicate, from WT and Spt5^dep MEFs comparing fold change (Spt5^dep/WT) versus the adjusted P‐value calculated using the DESeq2 package. The up‐ and down‐regulated genes indicated show a fold change of > 1.5 and P‐value < 0.05.

B. Splicing efficiency (spliced/total reads) calculated on merged mRNAseq data from three replicates of Spt5^dep and WT MEFs. A total of 185,149 splice sites were analyzed. The mean‐per‐junction splicing efficiency for all splice sites is plotted as scatter boxplots where 1 indicates complete splicing efficiency.

C. RT–qPCR for mRNA abundance in WT and Spt5^dep cells of three up‐regulated and three down‐regulated genes selected from mRNAseq differential gene expression analysis. Drosophila S2 cells were spiked in as in Fig 4B, and WT and Spt5^dep qPCR values were normalized against the Drosophila‐specific act5a transcript. The plot shows the mean of three independent WT and Spt5^dep samples with standard deviations.

D. Box plots showing the fold changes (Spt5^dep/WT) in GROseq gene body (TSS +500 bp to TTS) density and mRNAseq abundance upon Spt5 depletion. Fold changes are shown for all genes, genes significantly down‐regulated in Spt5^dep cells by > 1.5‐fold in mRNAseq (n = 223, P < 0.05) and genes significantly up‐regulated in Spt5^dep cells by > 1.5‐fold in mRNAseq (n = 368, P < 0.05). The P‐values were calculated using the unpaired t‐test.

E. Scatter dot plots showing the gene length distribution of > 1.5‐fold significantly up‐ or down‐regulated genes obtained from mRNAseq analysis. The red line marks the median of the distribution. Statistical significance was calculated by the Student's t‐test.