Fig5E_RACE_upstream-primer_R1_LG314.sgd

HEK293T ishXrn1 cells were treated with doxycycline for 3-4 days to induce knock down of Xrn1, then transfected with PA-X from the influenza A/Puerto Rico/8/1984 (H1N1) or vector, and luciferase reporters with 99bp inserted sequences from the STOML2 or YKT6 gene. Unlike other experiments in this collection, the insertion in this case was inside the intron of the luciferase reporter. RNA was collected 24 hrs later and analyze by 5' RACE using a primer that binds to luciferase sequences. In this case the position of the primer was such that the RACE would only pick up, if present, a 200 bp fragment originating from the inserted sequence.

将HEK293T ishXrn1细胞经多西环素（doxycycline）处理3~4天以诱导Xrn1基因敲低，随后分别转染甲型流感病毒A/波多黎各/8/1984（H1N1）来源的PA-X蛋白或空载体，以及携带STOML2或YKT6基因99bp插入序列的荧光素酶报告基因。与本数据集内的其他实验不同，本次实验的插入序列位于荧光素酶报告基因的内含子内部。收集转染24小时后的RNA，使用结合荧光素酶序列的引物通过5' 快速扩增cDNA末端（5' RACE）进行分析；本次实验中引物的设计位置使得，若存在源自插入序列的扩增产物，仅能得到长度为200bp的特异性片段。