As detailed in a previous post, chemically-modifying oligonucleotide adapters is an effective means to prevent adapter dimer formation during small RNA library prep. Just as primer dimers form when very little template DNA is used for PCR, adapter dimers can form with low starting concentrations of RNA. The CleanTag™ Ligation Kit for Small RNA Library Prep is a complete kit which is compatible with Illumina® technology that makes use of such modified adapters in optimized buffer conditions. The kit includes CleanTag™ chemically modified adapters that greatly reduce adapter dimer formation and is optimized for total RNA input from 1-1000 ng.
Overall, the kit contains 7 vials:
- CleanTag™ 3′ Adapter: PO DNA, 5′ (rApp) TGG AAT TCT CGG GTG CCA AGG (ddC) 3′
- CleanTag™ 5′ Adapter: PO RNA, 5′ GUU CAG AGU UCU ACA GUC CGA CGA UC 3′
- Enzymes 1 & 2 (2 vials)
- Buffers 1 & 2 (2 vials)
- RNase Inhibitor
- 3′ Adapter Ligation to RNA Template
- 5′ Adapter Ligation to Tagged RNA Template
- Reverse Transcription (RT) Reaction of Tagged RNA Library
- PCR Amplification of RT Product
- Magnetic Bead Purification
This ligation kit is used to perform the ligation step. For the RT and PCR amplification steps, users will also need Index Primers for Illumina® technology and high quality reagents and enzymes for reverse transcription and PCR, which can be purchased separately:
- 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)
*Do you want a DNA polymerase with the highest fidelity possible instead? In this case we recommend AccuStar™ DNA polymerase, which is similar to the Phusion® enzymes in fidelity and in the fact that it leaves blunt ends.
Many researchers have expressed interest in purchasing the CleanTag™ 3′ and 5′ Adapters separately for use in other kits and protocols. tebu-bio can supply essentially any chemically-modified oligonucleotide to European researchers, however the CleanTag™ Ligation Kit for Small Library Prep contains optimized reagents and buffers and involves an optimized protocol. For these reasons, users are strongly encouraged to use the complete kit.
One interesting note is that the modifications in the CleanTag™ adapters in conjunction with the high-salt, high-PEG concentrations in the buffer can cause Bioanalzyer® peaks to shift higher during analysis:
The miRNA peak should be at ~150 bp for crude samples and at ~140 bp when purified. piRNA peak should be at ~ 160 for crude samples. and at ~150 bp when purified.
Although the CleanTag™ Ligation Kit for Small RNA Library Preparation contains adapters compatible with the Illumina® sequencing platform, the tagged library can be converted into Ion Torrent™ compatible sequences during the PCR step.
Interested in learning more? Leave your questions or comments below!