title: GSM7332138: 21d Nkapl-WT eCLIP rep2; Mus musculus; RIP-Seq
library_construction_protocol: Fresh testes were collected from wild-type mice at P21, and the subsequent PBS suspension containing seminiferous tubules was crosslinked by UV-irradiation three times at 254 nm (400 mJ/cm2) and lysed in eCLIP lysis buffer with RNase I (LifeTech) and Turbo DNase (LifeTech). NKAPL-RNA complexes were immunoprecipitated with anti-NKAPL antibodies and protein A dynabeads. 2% of lysates were used as input and processed in parallel with immunoprecipitated samples. NKAPL-RNA complexes were dephosphorylated, and ligated with 3′ RNA adapter. NKAPL-RNA complexes were separated and transferred for RNA extraction. RNAs were reverse transcribed before stand-specific eCLIP library construction. RNAs were reverse transcribed with AffinityScript (Agilent) followed by treatment with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. The DNA adapter was ligated to the 3' end of cDNA with T4 RNA Ligase (NEB). Samples were cleaned up with MyOne Silane Dynabeads (Thermofisher) and subject to quantitative PCR to determine the appropriate number of PCR cycles. Libraries were amplified with Q5 Polymerase (NEB). Amplified PCR products were separated on 3% low-melting temperature agarose gel (Seakem GTG LMP), and size-selected libraries were purified with a MinElute gel extraction kit (Qiagen).