Ribosomal RNA (rRNA) accounts for ~90% of total cellular RNA. If your study is not researching rRNA, the abundance of these molecules can make it difficult to sequence RNA of interest. In eukaryotic organisms, the first step of RNA sequencing (RNA-seq) after RNA extraction is ribosomal depletion, which usually selectively captures the non-rRNA sequences by using oligonucleotides to anneal to the polyadenylated tails of messenger RNA (mRNA). While this method is useful for general RNA-seq of eukaryotic species, the mRNA of prokaryotes lacks the required polyadenylation. This type of rRNA depletion can also be insufficient for degraded DNA like that derived from formalin-fixed, paraffin-embedded (FFPE) samples. Ribosomal depletion of organisms like bacteria and viruses requires other methods.
Prezza et al. adapted a method called Depletion of Abundant Sequences by Hybridization (DASH) that involves DNA cleavage using CRISPR-associated Cas9. “Hybridization” in the method name refers to the hybridization of Cas9-bound guide RNA (gRNA) with target DNA. DASH uses in vitro Cas9 nuclease, directed by gRNA, to cleave and destroy high-abundance, unwanted cDNA sequences, while leaving desirable cDNA sequences intact. This study applies the DASH method to deplete bacterial cDNA derived from rRNA sequences in preparation for RNA-seq.
Prezza et al. isolated RNA from 2 bacterial species using TRIzol™ reagent. DNase I removed genomic DNA, and RNA quality was confirmed using an Agilent 2100 Bioanalyzer™ (Agilent Technologies). Target sequences of rRNA genes were chosen using a custom Python script (available on Github) and filtered based on GC content in addition to other criteria. The custom target sequences were ordered as oPools™ Oligo Pools from Integrated DNA Technologies, Inc., (IDT) and used to generate double-stranded DNA (dsDNA) templates. Single guide RNA (sgRNA) pools were derived from the dsDNA templates. Total RNA was used to generate cDNA before performing DASH, that cleaved rRNA-derived cDNA fragments. The resulting cDNA libraries were amplified to further enrich for the regions of interest and sequenced on Illumina systems before being processed and analyzed.
“We were very satisfied with our oPools Oligo Pools, especially for their fast turnaround time and high output amount. We also sequenced the gRNAs transcribed from the DNA template pool that we ordered and all of the expected sequences were there.”
—Dr Gianluca Prezza