Drosophila small RNA isolation v1

Drosophila small RNA sequencing samples are often overwhelmed by the abundant 30-nucleotide 2S rRNA fragment. Small RNA of interest can be isolated from total RNA without this fragment using gel size separation and a block oligo with sequence complementarity to the 2S rRNA fragment. In this protocol we detail the equipment, reagents and method for recovering small RNA (size range ~17-29 nucleotides) from total RNA using a urea-PAGE gel and ethanol precipitation, in preparation for deep sequencing. We draw on previous publications and available protocols to present step-by-step instructions with tips and tricks for successful small RNA isolation.