ExtFig6B_RACE_STOML2_R1_LG388_annotated.tif

HEK293T ishXrn1 cells were treated with doxycycline for 3-4 days to induce knock down of Xrn1, then transfected with a luciferase reporter with a 15 bp inserted sequence from the STOML2 gene, wild-type or containing mutations in GC basepairs to disrupt the hairpin structure, or contaning mutations in GC basepairs that preserve the hairpin structure. They were infected 24 hours later with influenza A/Puerto Rico/8/1984 (H1N1), either wild-type or PA-X deficient (deltaX) or mock infected. RNA was collected 24 hrs later and analyze by 5' RACE using a primer that binds to luciferase sequences.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STOML2基因15 bp插入序列的荧光素酶报告基因（luciferase reporter）载体，该载体包含三类变体：野生型、携带可破坏茎环结构的GC碱基对突变型，以及携带可保留茎环结构的GC碱基对突变型。转染24小时后，分别以野生型、PA-X缺陷型（ΔX）甲型流感病毒A/Puerto Rico/8/1984 (H1N1)进行感染，或设置模拟感染（mock infection）对照组。于感染后24小时收集RNA，使用结合荧光素酶序列的引物开展5'末端快速扩增（5' RACE）分析。