PAR-CLIP analysis of Drosophila Exportin-5 in S2 cells

S2 cells expressing FLAG-tagged Exportin-5 were labeled with 4sU and UV-crosslinked. Cells were lysed in RIPA buffer, and the lysate was treated with RNase, followed by immunoprecipitation by the FLAG-anibody to purify the RNA/Exportin-5 complex. RNA was extracted from the immunoprecipitate after protein digestion by Proteinase K, and subjected to small RNA sequencing library construction.

表达带FLAG标签的输出蛋白5（Exportin-5）的S2细胞经4-硫尿核苷（4sU）标记并进行紫外交联。将细胞于RIPA缓冲液中裂解，裂解液经核糖核酸酶（RNase）处理后，通过FLAG抗体进行免疫沉淀，以纯化RNA/输出蛋白5复合物。经蛋白酶K（Proteinase K）消化蛋白后，从免疫沉淀产物中提取RNA，并进行小RNA测序文库构建。