Figure 1

Adaptation 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.