title: Aichi/1968 NP mutant virus library (replicate 1) passaged in MDCK cells treated with 100nM 17AAG in 0.1% DMSO (Hsp90i) at 37 C for 48 h
design: 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.