70 ti fixed angle rotor

For the preparation of lentiviruses, the lentiviral shRNA expression plasmids were co-transfected into HEK 293T cells with pMD2.G envelope plasmid (Addgene #12259) and psPAX2 packaging plasmid (Addgene #12260) (gifts from Didier Trono) using FuGENE^® HD Transfection Reagent (Promega Corporation, Madison, WI, USA) following the manufacturer's protocols. The viral supernatants were collected after 48, 60 and 72 h, pooled, and concentrated by ultracentrifugation at 20,000 rpm for 2 h at 4 °C in a 70 Ti fixed angle rotor (Beckman Coulter, Palo Alto, CA). Aliquots of the viruses were frozen at − 80 °C until use.

Six samples from four EAV infected stallions were subjected to NGS. These were labelled as: EAVhucPL1(05/2013), EAVhucPL2(01/2009), EAVhucPL2(05/2013), EAVhucPL3(01/2009), EAVhucPL3(12/2012) and EAVhucPL4(05/2013) where letters were used for identification of the stallion and numbers in bracket represented month and year of sampling. Stallions hucPL2 and hucPL3 were positive for EAV RNA in their semen since at least 2006^{6 (link)} whereas stallions hucPL1 and hucPL4 tested EAV positive for the first time in 2013. Prior to sequencing, all samples were concentrated by ultracentrifugation. Briefly, 6 mL of seminal plasma was diluted five times in phosphate buffered saline pH 7.3 to 7.5 (PBS) and centrifuged at 3,000 × g for 10 min. Supernatant was then layered onto a 30% glycerol cushion (5 mL) in ultraclear thinwall polypropylene ultracentrifuge tube (Beckman). Following addition of PBS to the final volume of 35 mL, each sample was centrifuged for 2 h at 236,000 × g at 4 °C in a 70Ti fixed angle rotor (Beckman). Supernatant was removed and the pellet was resuspended in 0.5 mL of PBS. Viral RNA for NGS was extracted from this preparation using the same procedure as described above for RT-qPCR. Next generation sequencing was performed at Genomed SA using Illumina MiSeq Personal Sequencer.

To concentrate and purify proteins in a higher density complex such as a VLP, the samples were pelleted through a 20% sucrose in a 120 mM HEPES cushion at 100,000× g for 2 h at 4 °C with 45 Ti or a 70 Ti fixed-angle rotor (Beckman-Coulter, Brea, CA, USA), depending on the volume of the sample. After centrifugation, the supernatants were removed, the ultracentrifuge tubes were inverted for 5 min on a paper towel to remove the remaining supernatant, and the pellets were gently resuspended in a TNE buffer overnight at 4 °C, and an equal volume was processed for loading onto a density gradient, western blotting, or nanoLuciferase activity experiments.