Generating a Library from 28 nt RNAs...

[Carlo]

Hello all. I'm trying to generate RNA-Seq libraries suitable for use on the Illumina HiSeq 2000 instrument from purified 28 nt RNA fragments that have been generated by nuclease digestion. Nuclease digestion should produce fragments that are 3'-P and 5'-OH. I know that the Illumina small-RNA kit requires that fragments be 3'-OH and 5'-P (as miRNAs are). I was wondering if anyone had any suggestions as to an appropriate kit or protocol that I could use?

Alternatively, I thought that it may be possible to treat my short RNAs with T4 polynucleotide kinase (removing the 3'-P and phosphorylating the 5'-OH) so that they're suitable for use with the small-RNA kit, but I'm worried that the yield/efficiency may be low.

Any suggestions or opinions would be greatly appreciated!

Carlo

[garethmorgan]

Hi Carlo.
Sounds like you might be doing a footprinting experiment. I'm trying to get that working at the moment. The protocol I use is, roughly:
purify RNA
dephosphorylate 3' end
ligate with T4 RNA ligase to IDT's small cloning RNA linker:

http://www.idtdna.com/pages/products/mirna/mirna-cloning-products

reverse transcribe with a primer complementary to the linker
circularise with Epicentre CircLigase
PCR to add ends for sequencing.

I use T4 PNK without ATP to dephosphorylate the RNA and it seems to work well. I don't know if it's important to have a 5' phosphate but I think probably not.
Hope that helps.