Figure 6

Elongation rates are not altered upon Spt5 depletion

A Experimental scheme for elongation rate measurements. Following 72 h of 4‐HT treatment, WT and Spt5^dep cells were treated with 2 μM Flavopiridol for 4 h. Flavopiridol was washed off, cells were transferred to drug‐free medium and aliquots were taken immediately (0 h) and at 5–10 min intervals for up to 2 h. Samples were then processed for pre‐mRNA RT–qPCR analysis.

B, C Elongation rate measurements for two long (B) and two short (C) genes in WT and Spt5^dep cells following Flavopiridol inhibition and release into drug‐free medium. The UCSC genome browser view of each gene is shown on the left with a zoom‐in view on the transition zone in the early TSS‐proximal gene body as highlighted with the black box. The corresponding pre‐mRNA RT–qPCR analysis is shown on the right. In the browser profile, WT and Spt5^dep tracks are overlaid in blue and red representing WT and Spt5^dep tracks respectively, and black representing the overlap between them. The location and distance of each qPCR primer pair from the TSS is indicated. The RT–qPCR data for each time point are normalized to the value of the untreated sample (= 1). The elongation rate is calculated using the time point at which the qPCR curves reach the value of the untreated sample (shown as a dotted line). Data represent three independent replicates showing the mean and standard deviation for each sample.

D Analysis of proximal elongation rate (for the region between the promoter‐proximal paused site and the first intron–exon junction analyzed by RT–qPCR) and distal elongation rate (for the region between the first and second intron–exon junctions analyzed by RT–qPCR) between WT and Spt5^dep cells. For the proximal rate, the eight long genes and the two short gene genes were analyzed (n = 10). For the distal rate, only the eight long genes were analyzed (n = 8). Data were analyzed as in Fig 6B and C. ns., not significant by the Student's t‐test.