prg-1 mutant replicate 2
Small RNA samples were first oxidized using sodium periodate to oxidize unmethylated species. Small RNAs were then 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).