title: GSM8631968: Anti-NSUN7 CLIP-Seq of WT mice (with UV) - control - WT2; Mus musculus; RIP-Seq
library_name: GSM8631968
library_construction_protocol: Testis were obtained from 30 mice of wild type and 30 HA-Nsun7 mice. Testis were decapsulated in PBS and irradiated twice with 400 mJ/sm2 UV (GS Gene Linker UV Chamber Biorad). After that seminiferous tubules were lysed in PXL buffer (0.1% SDS, 0.5% deoxycholate, 0.5% NP-40 in PBS without Ca and Mg) and exposed to 5 cycles of 8 seconds ultrasonication with 70% amplitude. Then samples were incubated with Rnase T1 0.01 u/μl (Thermofisher) for 10 minutes at 37 C with shaking 1000 rpm. For immunoprecipitation were used Pierce™ Anti-HA Magnetic Beads (Thermofisher). Before elution RNA-protein conjugates, which were bound to magnetic beads, were exposed ro dephosphorylation with СIAP (0,5 U/мкл) (2386334, Invitrogen) for 15 minutes at 37 C with shaking 1000 rpm, which was followed by phosphorylation with T4 PNK (Thermofisher) 30 minutes at 37 C with shaking 1000 rpm. RNA was eluted from the magnetic beads with proteinase K (Thermofisher) for 20 minutes at 37 C with shaking 1000 rpm. RNA libraries were prepared with the NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible, E7330S) in accordance with the manufacturer's recommendations, omitting the size-select step at the final stage of the protocol.