Depletion of TDP1 results in decrease of Pol I-derived transcripts. Total RNA was isolated after the induction of TDP1 RNAi for the indicated time, cDNA was made and qPCR performed to monitor transcript levels from various loci. ( A ) Schematic of T. brucei BF T3-TDP1 that stably expresses VSGT3 in the active ES, which is selected for with blasticidin (Blast). ES promoters are indicated with flags, relevant genes with boxes and transcription with an arrow. TDP1 RNAi is expressed from opposing tetracycline inducible T7 promoters (arrows). ( B ) Analysis of Pol II transcripts after induction of TDP1 RNAi with tetracycline (Tet) for the time indicated below. Transcripts analysed are: TDP1 , actin, γ-tubulin (γ-tub) and Pol I and precursor transcripts of α-tubulin and Pol I. Data shown in this panel are not normalized to the levels of another transcript, with the exception of the TDP1 transcript, which is normalized to actin. ( C ) Schematic of the rDNA locus. The promoter is indicated with a flag and various rRNA genes with boxes. Precursor rRNA transcripts as determined previously ( 34 ) are indicated below, where P1 is 3.4 kb, P2 is 5.8 kb and P3 is 5.0 kb. Regions analysed by qPCR are shown with lettered brackets. ( D ) Reduction in Pol I-derived transcripts after induction of TDP1 RNAi. Relative transcript amounts were quantitated with the primers indicated in panel C. Levels of rRNA precursor transcript or transcript from VSGT3 (in the active ES) or VSG121 (in a silent ES) were also monitored. Data shown have been normalized against values for the actin transcript shown in panel B. Relative transcript levels were calculated by dividing the value for the level of transcript at any given time point by the level present in uninduced (0 h) samples. Results are the mean of triplicate independent experiments, with standard deviation indicated with error bars.