GSM5493800: Ago2_pachytene_2_CLIPinput; Mus musculus; OTHER
Purified germ cell nuclei from adult mouse testes were crosslinked with 254nm UV light. Nuclei were lysed and sonicated in a Bioruptor sonicator (Diagenode) at 4C, 5 min, 30sec on/30sec off. The nuclear lysate was then digested with RNase I (AM2295, Thermo) and Turbo DNase (AM2239, Thermo), and immunoprecipitated using anti-AGO2 antibody (04-642, Millipore). Next, the protein-bound RNAs were ligated with 3' end adapters. Protein-RNA complexes were subjected to gel electrophoresis and transferred to a membrane, and RNA was isolated by cutting the membrane at the expected size range. Libraries were prepared as described in van Nostrand et al, 2016. cDNA was generated by reverse transcription, DNA adapter was ligated to the 3' end of the cDNA, and the library was amplified by PCR. Screening of subclones prior to high-throughput sequencing was performed for quality control.