Next generation sequencing has quickly become the preferred method over tiling arrays for most genomics and transcriptomics needs. The major exception has been the study of microRNAs, where highly sensitive probe arrays such as the 3D-Gene® miRNA profiling platform are still widely used. A large part of the reason for the persistence of array dominance in small RNA expression profiling is caused by the variability introduced in sequencing library prep protocols involving complicated hands-on PAGE purification steps.
The CleanTag™ Ligation Kit for Small RNA Library Preparation now allows users to remove the Gel Purification steps from their protocols and shift to more automated bead purification protocols. This is particularly important for cases when RNA quantity is limiting. Traditional small RNA library prep protocols will result in the formation of adapter dimers (similar to primer dimers) when RNA quantities are limiting, thus greatly reducing the number of usable reads.
The chemically-modified adapters used in the CleanTag™ protocol fail to form adapter dimers, eliminating the need for a PAGE purification step to purify the small RNA library. Depicted in the diagram below, standard adapters may result in direct ligation of adapters, while the chemical modifications in CleanTag™ adapters prevent this adapter dimer formation.
The CleanTag™ Ligation Kit for Small RNA Library Preparation is not a complete NGS library prep kit, however, so users can adapt it to their own protocols. Users will need to purchase separately Index Primers for Illumina® technology and high quality enzymes and reagents for reverse transcription and PCR, such as:
- Index Primer Set 1 (Primers 1-12 with RT and Forward PCR Primer)
- Index Primer Set 2 (Primers 13-24 with RT and Forward PCR Primer)
- Reverse Transcriptase (EpiScript™ Rnase H– RT)
- PCR 2X PreMix (FAILSAFE 2X PCR PREMIX E is routinely used with ScriptSeq™ Kits)*
- dNTPs (one 100 mM vial each of dATP, dCTP, dGTP and dTTP)
- RNAse Inhibitor (ScriptGuard™ RNase Inhibitor)
For a higher fidelity polymerase than the FAILSAFE 2X PCR PREMIX E, we recommend AccuStar™ DNA polymerase, which is similar to the Phusion® enzymes in fidelity and in the fact that it leaves blunt ends. Similarly, while EpiScript™ Rnase H– RT is a much less expensive alternative to SuperScript® II, MonsterScript RT Kit (EpiCentre’s answer to SuperScript® III) might be preferred.
To replace the PAGE gel cleanup step in traditional miRNA library prep protocols, one can opt for bead purification. Beckman’s Agencourt AMPure XP – PCR Purification is probably the most familiar to users, however some smaller companies offer similar products such as the HighPrep™ PCR (MagBio Genomics). These systems tend to be magnetic beads carrying modifications that make them able to bind DNA. This patent suggests that one can use standard BioMag® Dextran-coated Charcoal for PCR clean-up and some of the original work from the Whitehead suggests simple BioMag® Carboxyl beads can be used for what they called “solid-phase reversible immobilization of PCR products.
Any questions about the CleanTag™ Ligation Kit for Small RNA Library Preparation? Leave your comments below!