Figure 1From: 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cellsAdaptation of the DRB assay for measuring elongation rates in short time scales by RNA sequencing. (A) Analysis of OPA1 and TTC17 pre-RNA in HeLa cells, immediately after 3 h DRB treatment (DRB) and at the indicated time points after DRB removal. NT = non-treated. Pre-mRNA levels were determined by qRT-PCR, employing intronic primers. All values were normalized to 18S RNA in the same sample. Levels in non treated cells were set as 1. Bars indicate averages of three independent experiments; error bars represent standard deviation. (B) Relative pre-mRNA/mRNA ratios in total RNA (CON) and 4sU-enriched RNA (4sU). HeLa cells were labeled for 8 min with 4sU (1mM). RNA was isolated, biotinylated, and enriched on streptavidin beads. qRT-PCR analysis of OPA1 and TTC17 pre-mRNA and mRNA was performed on the enriched RNA, as well as on total (non-enriched) RNA. The pre-mRNA/mRNA ratio for each gene in the CON sample was arbitrarily defined as 1.0. (C) Scheme of the timing of DRB and 4sU treatments in the different conditions. NT = non-treated, DRB = 3 h of DRB only, 4 min = 3 h of DRB and harvesting 4 min after DRB removal, 8 min = 3 h of DRB and harvesting 8 min after DRB removal. Red squares = 4sU, orange stars = biotin. Following the indicated treatments, isolated 4sU-labeled RNA was biotinylated and purified with magnetic streptavidin beads as described in [42] and subjected to high throughput sequencing.Back to article page