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
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
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