RNAseq of of piRNA reporter strain bearing silent mutation In 21UR-4864 target site on w03h9.2 gene
Small RNA samples were first treated with a recombinant 5' polyposhatase to remove the g and b phosphates from 5' triphosphorylated RNAs. Following phosphatase treatment small RNAs were ligated to 3' adaptors using T4 RNA ligase 2  in the presence of 25% PEG8000 (NEB)  at 15 C overnight. 5' adaptors were then ligated to the product using T4 RNA ligase 1 at 15 C overnight. Ligation products were converted to cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). cDNAs were then amplified by PCR, libraries were sequenced on the Illumina Novaseq platform using the SP100 flowcell (SP 2 X 50 bp).