Antifoam a concentrate

Each experiment was conducted in triplicate. A volume of 35 ml of phage buffer 1X (20 mM tris, 100 mM NaCl and 10 mM MgSO₄) containing between 4.4 X 10⁶ and 1 X 10⁸ plaque forming units per millilitre (PFU/ml) of each phage and 5 μl of Antifoam A concentrate (Sigma-Aldrich, St-Louis, USA) was placed in a 6-jet Collison (BGI, Waltham, USA) supplied with 20 psi of compressed air (medical grade) and nebulized for 10 minutes. For MNV-1, 30 ml of viral stock (between 3.3 X 10⁵ and 4.4 X 10⁶ PFU/ml) that had been frozen at -80°C were thawed and placed in the nebulizer. Aerosols were forced through diffusion dryers of different lengths (327.4 cm, 203.7 cm, 37.7 cm) before they entered the chamber, in order to control the RH and achieve 20%, 55% and 85%, respectively. The particles generated had a mass median aerodynamic diameter (MMAD) of 1.10 ± 0.03 μm at 20% RH, 1.27 ± 0.03 μm at 55% RH and 1.24 ± 0.04 μm at 85% RH. The targeted temperature inside the rotating drum was 19°C ± 1°C. Aerosols were mixed for 10 minutes in the drum to achieve an even distribution before particles were counted using an APS. Ozone or air (reference condition) was injected into the chamber, and the aerosols were in contact with the gas for 0, 30 or 60 minutes before the air was sampled.

Each sample of 10,000 J2s was suspended in 650 μl of modified lysis buffer RLT Plus (Qiagen, Hildren, Germany) (1% v/v β-Mercaptoethanol (Sigma Aldrich, St. Louis, MO, United States), 0.2% v/v Antifoam A concentrate (Sigma Aldrich, St. Louis, MO, United States), 20 U/ml SUPERaseIN (Invitrogen, Carlsbad, CA, United States), 20 μg/ml Proteinase K (Invitrogen, Carlsbad, CA, United States). The nematodes were disrupted with one 6 mm Grinding Satellites (OPS Diagnostics, Lebanon, NJ, United States) and 200 μL of 1 mm zirconium beads (OPS Diagnostics, Lebanon, NJ, United States) in 2 ml tubes using a Biospec 3110Bx Mini-Beadbeater-177 1 (BioSpec Products, Bartlesville, OK, United States) at 4,800 oscillations/min for three 20 s intervals. Total RNA was extracted using RNeasy Mini Kit Plus (Qiagen, Hildren, Germany) according to the manufacturer’s instruction and stored at −80°C. RNA integrity assessment and sequencing were carried out at the GENEWIZ sequencing facility (South Plainfield, NJ, United States). The RNA samples with a RNA integrity number (RIN) value of ≥7 were used for the library preparation. The libraries were generated using TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, United States) and a paired-end (2 × 150 bp) sequencing was done using the HiSeq2500 (Illumina, San Diego, CA, United States).