Total RNA sequencing, or rRNA depletion sequencing, enables the analysis of both coding and noncoding transcripts leading to high confidence observations of features such as allele-specific expression, alternative splicing, and novel transcripts. This approach relies on rRNA depletion to remove off-target transcripts. Illumina’s rRNA depletion kits enzymatically degrade the ribosomal sequences before cDNA synthesis, with oligonucleotide probes used to flag the rRNA content. This negative selection leaves behind all other transcripts for sequencing and is designed for a higher tolerance of lower-quality RNA. It results in increased ribosomal content and a larger proportion of intronic sequencing from nascent transcripts. As this protocol requires homologous probes and high RNA integrity for adequate depletion, it can be less efficient than polyA tail capture depending on probe specificity; however, it is irreplaceable for organisms lacking polyA tailed transcripts and for reliably examining noncoding transcripts.
For rRNA depletion sequencing, SeqCenter uses Illumina’s Stranded Total RNA Prep with Ribo-Zero Plus Microbiome kit to generate unbiased high-quality RNA libraries. The two depletion kits included in this prep are targeted towards major model organisms as well as a broader range of bacteria with clinical significance. Beyond that, SeqCenter is able to design custom depletion probes in partnership with Illumina to ensure optimal depletion in your sample if your project’s organism is not covered within the standard range. For more information, see the depletion section below.
We strongly recommend visiting our overall Illumina RNA services page for additional considerations when planning any RNA experiment and encourage all customers to check out the tips for handling RNA and guidance when assessing sample quality for submission.