Aichi/1968 NP wildtype virus passaged in MDCK cells at 37 C for 48 h (replicate 3)
Two plasmid libraries were used to generate two biological replicate viral libraries, one of which was used twice to perform two technical replicates of the deep mutational scanning. MDCKdn-cHSF1 cells were plated in 15 cm plates at a density of 6  10^6 cells/dish, and treated with 0.1% DMSO or 1 mg/ml doxycycline for 24 h at either 37 C or 39 C. Deep mutational scanning was also performed in cells treated with an Hsp90 inhibitor (100 nM 17-AAG; 90 min. pre-treatment). 5  10^6 infectious virions (as determined by a TCID50 assay) from each viral library were used to infect four 15 cm plates from each condition at an MOI of 0.1 (the cells expanded to ~12.5  10^6 cells per plate by the time of infection). 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. For infection, the cellular growth media was replaced with WSN media containing a mutant virus library, wild-type virus, or no virus for mock infection. After 2 h, the inoculum was replaced with fresh WSN media containing 0.1% DMSO, 1 mg/mL doxycycline, or 100 nM 17-AAG. 48 h post-infection, the viral supernatant was harvested, centrifuged at 1000  g for 5 min to remove cell debris, and stored at 80 C. Viral RNA was extracted from the infectious supernatant using a Viral RNA Mini Kit (Qiagen) and reverse transcribed using the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Agilent). At least 10^6 NP molecules were PCR-amplified for preparation of the sequencing libraries, as previously described (as in Doud et al. [Viruses, 2016. 8(6): p. 155]). The resulting amplicons were sequenced on an Illumina HiSeq 2500 in rapid run mode with 250  250 bp paired-end reads.
wt_37C_replicate3