This protocol is for performing Depletion of Abundant Sequences by Hybridization (DASH) after preparing sequencing libraries and pooling together.DASH is most useful for RNA-seq of human metagenomic samples, where abundant species such as human mitochondrial ribosomal RNAs (rRNAs) occupy a majority of the sequencing space available, leaving a minor fraction for regions of interest. DASH treatment is performed after ligation of adapters and unique barcoding of the RNA-seq library. It employs CRISPR-Cas9 complexed to a set of guide RNAs (gRNAs) targeted to the abundant regions to be depleted in a given library. These abundant regions in the library are then cleaved, leaving only the fragments with intact adapters on both ends to be further amplified and sequenced.