replicate-2_DMSO39
Biological replicate viral libraries were independently generated from plasmid libraries (as in Doud et al. [Viruses, 2016. 8(6): p. 155]). Relevant expression constructs in HEK293ATF6f/XBP1s cells were induced 8h after plating at either 37 ?C or 39 ?C, and 16h later 1  10^6 Infectious virions from each viral library were used to infect two 15 cm plates in each condition at an MOI of 0.01 virions/cell by incubation with virus-containing WSN medium for 2h. In addition, one 15 cm plate at both 37 ?C and 39 ?C was either mock infected (negative control) or infected with wild-type virus. After 2 h, the inoculum was replaced with fresh WSN media containing 0.01% DMSO, 0.1 ug/mL doxycycline, or 0.1 ug/mL doxycycline and 1 uM TMP. 48 h post-infection, the viral supernatant was harvested, cleared by centrifugation and virions were concentrated by ultracentrifugation, followed by decantation.Virion pellet was resuspended in 140 uL QIAgen Viral RNA Buffer AVL. Viral RNA was then extracted using a QIAgen Viral RNA mini kit, per the manufacturers instructions, changing collection tubes at each step. Viral RNA was reverse transcribed using the AccuScript High Fidelity 1st strand cDNA synthesis kit (Agilent) using HA-specific primers. At least 10^6 HA molecules were PCR-amplified for preparation of subamplicon sequencing libraries, as previously described [Doud, op. cit.]. The resulting libraries were sequenced on an Illumina HiSeq 2500 in rapid run mode with 250  250 bp paired-end reads.