Fig6I_PCR_A549_R2_LG402_annotated.pdf

RT-PCR analysis of viral RNA to verify presence of inserted sequences. A 51 bp segment of the STOML2 gene was inserted in the M segment of A/Puerto Rico/8/1984 (H1N1) ("PR8"). Viral RNA was extracted from viral stocks (G), supernatants from MDCK cells 48 hours post infection (H), or Xrn1 knock-out A549 cells 16 hours post-infection. Viruses analyzes included  viruses harboring no insert, inserted WT STOML2 or mutant STOML2 sequences that is not cut by PA-X, either in the WT PR8 or PR8-PA(∆X) background. The RNA was then reverse-transcribed to cDNA and PCR amplified using primers located on either side of the STOML2 sequence to visualize on an agarose gel whether the STOML2 sequence was retained.