Selective (custom) transcript depletion enables the removal of user-defined transcripts from RNA samples prior to cDNA synthesis. The depletion ribosomal RNA (rRNA) and highly abundant mRNAs (e.g., globin from blood samples), improves the sensitivity and economy of RNA-Seq, as fewer sequencing reads are associated with “unwanted” transcripts. This enables the detection of more rare transcripts and subtle fold-changes in gene expression levels between biological or experimental conditions from a given amount of sequencing. Alternatively, it allows for less sequencing to be performed to achieve experimental goals.
Approaches and Techniques
Highly expressed RNA transcripts, including rRNA, globin and/or others, are depleted using DNA oligos that are complementary and bind to rRNA transcripts. After hybridization, RNA:DNA hybrids are enzymatically removed using RNase H followed by cDNA synthesis and RNA library preparation.
KAPA RNA HyperPrep Kits with RiboErase (HMR) or RiboErase (HMR) Globin provide for the selective depletion of transcripts (e.g., rRNA, globin mRNA and/or other highly abundant mRNA species), followed by cDNA synthesis and library construction. The enzymatic-depletion chemistry is robust, compatible with both high-quality and degraded inputs (such as FFPE) from all organisms.
Depletion workflows provide a complete view of the transcriptome, while minimizing the number of wasted sequencing reads from “uninteresting” transcripts.