Cellular and mitochondrial ribosomal RNAs (rRNA) represent 80 – 90 % of total RNA in eukaryotic cells, and the efficiency and sensitivity of high-throughput RNA sequencing is significantly affected by this high content of identical sequences. To prevent sequencing these unwanted rRNAs, poly(A) selection can be employed, which enriches for mRNAs and polyadenylated ncRNAs. However, this approach might result in 3’ end bias and is blind for the non-polyadenylated part of the transcriptome. The alternative approach, depletion of rRNA in total RNA samples, enables the efficient detection of mRNAs, ncRNAs, sRNAs, miRNAs, and many more biotypes (O’Neil et al. 2013).
Lexogen’s RiboCop rRNA Depletion Kit, initially released in 2016, allows efficient removal of cytoplasmic 28S, 18S, 5.8S, 45S, 5S, as well as mitochondrial mt16S, mt12S ribosomal RNA sequences in human, mouse and rat samples. The kit was also successfully tested on hamster, mini pig, and zebrafish samples. The high depletion efficiency of the RiboCop method was recently validated with only 0.3 – 1.5 % rRNA reads remaining in human total RNA after depletion (Vecera et al. 2019).
RiboCop can be fully automated and coupled with any downstream method requiring rRNA depletion such as Lexogen’s CORALL Total RNA-Seq Library Prep Kit for whole transcriptome analysis, Poly-Seq, and Ribo-Seq. RiboCop works with input RNA in a range from 1 – 1,000 ng total RNA and with intact or degraded (such as FFPE) samples.
Retaining this efficiency and all applications, Lexogen has further optimized the workflow of the RiboCop rRNA Depletion (Human/Mouse/Rat) Kit: the new version 1.3 protocol features fewer protocol steps, reduced hands-on time (30 minutes), and a shorter overall protocol time (1.5 hours). All supplied RiboCop Kits (including the recently distributed V1.2 kits) are compatible with the new V1.3 protocol.
While the protocol has changed, pricing, Cat. No., and the physical kit layout remain the same, and users are still able to follow the V1.2 protocol, if they wish to. For further information visit the RiboCop product page and check out the latest RiboCop-citing publications.
O’Neil, Dominic; Glowatz, Heike; Schlumpberger, Martin (2013): Ribosomal RNA depletion for efficient use of RNA-seq capacity. In: Current Protocols in Molecular Biology Chapter 4, Unit 4.19. DOI: 10.1002/0471142727.mb0419s103.
Vecera, Marek; Sana, Jiri; Oppelt, Jan; Tichy, Boris; Alena, Kopkova; Lipina, Radim et al. (2019): Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs. In: PLoS One 14 (2), e0211978. DOI: 10.1371/journal.pone.0211978.