Figure 4.

qDRIP allows for effective normalization when R-loops are acutely perturbed by transcription inhibition. (A) Genome browser view taken from a long gene (AUH) showing effects of DRB on hybrid formation. In order from top to bottom, tracks shown are two biological replicate DRB-treated IP samples, two biological replicate control (DMSO) IP samples, and a track showing genes. All tracks are normalized by reads per million mapped. Red indicates negative strand signal, while blue indicates positive strand signal. Bent arrows represent TSS, while large triangular arrows represent TES. (B) Scatter plots showing read-counts over Pol I or Pol III-transcribed regions compared for DMSO and DRB treatment using read counts to normalize (left) and spike-ins to normalize (right). Individual regions are shown as grey dots, while the regression line and bootstrapped 95% CI are shown as a grey line and grey band, respectively. Blue diagonal line represents expected trend line if read counts are equal. The normalization factor calculated using read counts is 1.373 with a bootstrapped 95% CI of (1.247, 1.459), while the normalization factor calculated using spike-ins is 1.170 with a bootstrapped 95% CI of (1.063, 1.242). Altogether, spike-ins significantly reduced overestimation of non-pol II genes (P = 0.006, non-parametric bootstrap of the difference of means). (C) Metaplots of DMSO (blue) and DRB (red) IP signal over the first 200 kb of all genes longer than 200 kb expressed in HeLa cells by GRO-seq (36), normalized using total read counts (left) or spike-in read counts (right).