MILI-IP RIP-Seq of Miwi[+/RK]; replicate 2
Radiolabeled piRNAs were purified with 15% UREA-PAGE and ligated to a modified miRCat 3 Linker-1 (IDT) containing eight extra random nucleotides at the 5-end. Ligated RNA was reverse-transcribed by a 5 phosphorylated long primer containing 3 and 5 adapter complementary sequences. The produced cDNA was circularized and amplified by 25 PCR cycles with adapter specific primers carrying p5 and p7 Illumina flow cell binding sequences.