ExtFig3E_RACE_INSIG1_SLC7A5_R1_LG305_annotated.tiff

HEK293T ishXrn1 cells were treated with doxycycline for 3-4 days to induce knock down of Xrn1, then transfected with the luciferase reporters containing 99 bp insertions from the BCAP1, INSIG1, SLC7A5, STOML2, YKT6, and TUBA1B genes, and where indicated, with PR8 PA-X. RNA was extracted and used to run 5’ RACE with primers annealing to luciferase sequences. DNA bands were purified and sequenced to confirm their identities

将HEK293T ishXrn1细胞经多西环素处理3~4天以诱导Xrn1基因敲低，随后转染携带BCAP1、INSIG1、SLC7A5、STOML2、YKT6及TUBA1B基因来源的99 bp插入片段的荧光素酶报告质粒；在指定实验组中，一并转染PR8 PA-X。提取细胞总RNA，以其为模板，使用靶向荧光素酶序列的引物进行5' 末端快速扩增（5’ RACE）。纯化所得DNA条带并进行测序，以验证其序列身份。