Reduction of rRNA during sRNA library construction using a complementary blocking oligo, in Drosophila melanogaster

This experiment was designed to improve the percentage of miRNA mapping reads from sRNA library sequencing, by reducing the amount of one particularly abundant rRNA 30mer sequence. We extracted the sRNA fraction from 50 pooled male D. melanogaster abdomens, and constructed 2 cDNA libraries from the resultant RNA. For one library (blocked), we introduced a 30nt blocking oligo, with a sequence complentary to the most abundant contaminating rRNA fragment, with the aim of preventing ligation of that rRNA to library adapters. The other library (unblocked) was constructed according to standard protocol. Both libraries were sequenced using a HighSeq2500. We extracted the sRNA fraction from 50 pooled male D. melanogaster accessory glands and testes and constructed 2 cDNA libraries from the resultant RNA. For one library (blocked), we introduced a 30nt blocking oligo, with a sequence complentary to the most abundant contaminating rRNA fragment, with the aim of preventing ligation of that rRNA to library adapters. The other library (unblocked) was constructed according to standard protocol. Both libraries were sequenced using a HighSeq2500.