Extended Data Fig. 7: RO does not reduce large gene transcription.
A, B: Exponentially growing cells were grown for 16h in different media (colour code shown on the right). The map of the genes is shown above the histograms with the position exons (ex) (like in Extended Data Fig. 5a) and of intronic primers used for the qPCR (i-F/R) and A: Fixed chromatin from JEFF lymphoblasts was immunoprecipitated with anti-RNA polymerase II antibodies (Methods). The density of RNA polymerase II along FHIT and WWOX was determined by qPCR with the indicated intronic primers. Results are presented as fold enrichment relative to the levels obtained with control immunoglobulins. Note the accumulation of RNA polymerase on the FHIT promoter detected by the intron 1 (i1) primers. Two biologically independent experiments were done with similar results. B: Comparison of nascent RNA levels along the FHIT and WWOX in JEFF lymphoblasts, and of primary RNA levels along the NEGR1 in MRC5 fibroblasts. JEFF cells were treated as in A and pulse-labelled with 5-ethynyl-uridine. Nascent RNAs were purified by affinity chromatography and quantified by RT-qPCR with the indicated primers. Results are presented relative to the level in NT cells. Experiments were done once. C: FHIT transcription across the cell cycle. Non-treated JEFF lymphoblasts were FACS-sorted (left inset) in 4 fractions and RNA extracted from each of them. Primary RNA levels were quantified by RT-qPCR as in A. Results for each primer pair were normalized using cyclophilin B (PPIB) RNA as endogenous control and are presented relative to the total amount of transcripts (sum of G1+S1+S2+G2/M). In the rightmost histogram, the sum of the transcript levels detected by all primer pairs in each fraction is expressed relative to the sum of the levels of all transcripts in all fractions. Two biologically independent experiments were done. Data are presented as mean values.
