Accurate monitoring of genome transcription activities is of crucial interest for deciphering gene expression and for a better understanding of RNA biology. Over the past years, various experimental methods for RNA Pol II mapping density across the whole genome have been designed. Here, I’d like to offer a brief introduction to the human Native Elongating Transcript-Sequencing (NET-Seq) method.
Any researcher who has purchased a complex custom oligonucleotide with chemically-modified bases has probably noticed that prices drop dramatically with increased production scale. Also, companies are reluctant to promise a final yield and prefer, rather, to issue quotes for initial starting scales.
For example, a request for custom synthesis of the modified preadenylated linker oligo below with a 5′ Adenylate and a 2′,3′-Dideoxycytidine blocking group to prevent self ligation might yield quotes for 1.0 umole starting synthesis scale (1 OD guaranteed yield) or 15 times the amount (15 OD guaranteed yield) for only about 4 times the cost.
Preadenylated linker oligo: 5′- (rApp) AGA TCG GAA GAG CGG TTC AG (ddC) -3′
In the case of this particular oligo, the 5′ Adenylate group is useful for protocols that involve the ligation to the 3′ end of RNA libraries using T4 RNA Ligase Deletion Mutant 2, however 5′ adenylated oligos require an extra purification after the adenylation step and are not compatible with heat-sensitive modifications. Consequently, PAGE purification is required leading to increased costs, particularly for small production scales.
Structure of 5′ Adenylate:
But what if researchers could get together and order a bulk purchase of commonly used oligos and benefit from the relatively low costs of bulk production?
For the oligonucleotide above, we tested this idea by contacting multiple RNA biology groups throughout Europe performing a technique called iCLIP (individual-nucleotide resolution Cross-Linking and ImmunoPrecipitation). Developed by Jernej Ule, the next generation sequencing-based technique is used to identify specific RNA sequences bound by RNA binding proteins. Overall, the response was positive and the researchers liked the idea of sharing a single, high quality oligonucleotide production. Many indicated that the oligonucleotide called L3 seemed unnecessarily expensive due to the custom production. They also liked the idea that one researcher could quality control the oligonucleotide and others would have access to the same pre-validated production lot. We therefore launched production and created a new catalog product 3′ iCLIP Primer, and the first users have used this new product with positive results. User data provided by Jernej Ule’s former student Zhen Wang (Laboratory of Hervé Le Hir, Institut de Biologie de l’École Normale Supérieure) demonstrates that the oligonucleotide will perform well in iCLIP protocols:
Figure 1. 15% TBE-Urea gel of RNAs ligated with L3 linkers. A synthetic RNA of 40nt was used, and the ligation was performed with T4 RNA Ligase at room temperature for 2.5 h. Different PEG conditions were tested for ligation efficiency, and the old L3 linker was used as a control. In each reaction, 10pmol of RNA and 20pmol of linker was used in a total of 20ul. Gel visualized with a SYBR®-based stain such as GreenView™ Plus DNA Gel Stain.
European scientists interested in custom production of complex oligonucleotides can contact tebu-bio using this form.
Let us know if you think that your oligonucleotide has the potential to be our next catalog product!
New!: We’ve also just released TruSeq™ compatible Index Primers 1-12 and 13-24 including RT and Forward PCR Primer for our sequencing customers, intended for use with the new CleanTag™ Ligation Kit for Small RNA Library Prep.
Legal notes: SYBR® is a registered trademark of Life Technologies. TruSeq™ is a trademark of Illumina.
Individual-nucleotide resolution UV CrossLinking and ImmunoPrecipitation (iCLIP) enables the identification of protein-RNA interactions. The original CLIP protocol described by Jernej Ule et al. in 2003 (1) has evolved significantly (2, 3), and the current iteration of the iCLIP protocol requires a core set of oligonucleotides and enzymes.