Fig. 2
Depletion of Abundant Sequences by Hybridization (DASH) targeting abundant mitochondrial ribosomal RNA in HeLa RNA extractions. a Normalized coverage plots showing alignment to the full-length human mitochondrial chromosome. Before treatment, three distinct peaks representing the 12S and 16S ribosomal subunits characteristically account for a large majority of the coverage (>60 % of total mapped reads). After treatment, the peaks are virtually eliminated — with 12S and 16S signatures reduced 1000-fold to 0.055 % of mapped reads. b Coverage plot of Cas9-targeted region with 12S and 16S gene boundaries across the top. Each red arrowhead represents one sgRNA target site. We chose 54 target sites, spaced approximately 50 bp apart. c Scatterplot of the log of fragments per kilobase of transcript per million mapped reads (log-fpkm) values per human gene in the control versus treated samples illustrate the significant reduction in reads mapping to the targeted 12S and 16S genes. DASH treatment results in 82- and 105-fold reductions in coverage for the 12S and 16S subunits, respectively. The slope of the regression line (red) fit to the untargeted genes indicates a 2.38-fold enrichment in reads mapped to untargeted transcripts. R-squared (R 2) value of the regression line (0.979) indicates minimal off-target depletion. Between replicates, the R2 coefficient between fpkm values across all genes is 0.994, indicating high reproducibility (three replicates). Notably, one gene, MT-RNR2-L12 (MT-RNR2-like pseudogene), shows significant depletion in the DASHed samples compared with the control