Picture from Duan, Ye et al. (2020) MicroPubl Biol

Figure 1: A. Protocol for depletion of ribosomal RNAs (rRNA) from samples of C. elegans RNA. Total RNA was extracted from worm lysates, heat denatured to melt secondary structure, and hybridized to a molar excess of the DNA antisense oligo (ASO) mixture. rRNA annealed to their complementary oligonucleotides were digested by thermostable RNase H, followed by removal of genomic DNA and ASOs with DNase. The reaction was then passed through a size selection column to desalt the sample and remove the truncated rRNA fragments and small RNAs less than 150 nt (e.g., tRNAs), and the RNA was cloned and sequenced. B. Alignments of ASOs to C. elegans 26S, 18S, 5.8S and 5S ribosome RNAs (rRNA). Some ASOs have complementarity to both a 5' segment and a 3' segment of 5.8S sequence. Images are primarily generated by SnapGene Viewer 5.1.4.1. C. Sequencing read coverage (linear scale; image generated by IGV 2.7.2 (Robinson et al., 2011)) at the Y59C2A.2 and C50C3.2 mRNA loci, corresponding to the top two predicted off-target sites of ASOs (red bar; see text for discussion). Note that the off-target depletion of mRNA sequence appears to be local and does not appreciably impact read density in other regions of those genes. Reads were from 4 libraries of different genotypes. D. Sequencing read coverage (linear scale) at the C. elegans primary rDNA locus from 4 libraries of different genotypes. We suggest that the reads flanking the rRNA come from primary rRNA transcripts, which were not targeted by ASOs (Ellis et al., 1986). E. Biotype proportions of reads from 12 RNA-seq libraries of 4 different genotypes prepared based on the rRNA depletion. F. Biotype proportions of reads in (E) after manually removal of signal recognition particle (srpR) RNA reads, which are predominantly enriched. The lower panel shows the proportions of the cytoplasmic rRNA reads (with ASOs) and mitochondrial rRNA reads (without ASOs) among the rRNA reads.