iCLIP of eIF4A3 and PTB in HeLa cells. iCLIP of eIF4A3 and PTB in HeLa cells

HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37°C. For iCLIP, HeLa cells expressing eIF4A3-GFP or PTB-GFP were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries for eIF4A3 and PTB were sequenced with 50 bp on an Illumina HiSeq instrument.

HeLa细胞（HeLa cells）于添加10%（体积比）胎牛血清（FCS）、青霉素/链霉素的杜尔贝科改良伊格尔培养基（DMEM）中，于37℃、5%二氧化碳（CO₂）环境下进行培养。针对紫外交联免疫沉淀测序（iCLIP）实验，将表达eIF4A3-绿色荧光蛋白（eIF4A3-GFP）或PTB-绿色荧光蛋白（PTB-GFP）的HeLa细胞用多西环素（doxycycline）诱导，使重组蛋白水平与内源性同源蛋白水平匹配，随后以150 mJ/cm²、254 nm的紫外线（UV light）进行照射。针对eIF4A3与PTB的iCLIP cDNA文库，采用Illumina HiSeq测序仪（Illumina HiSeq）完成读长为50 bp的测序。