Superscript 3 one step rt pcr with platinum taq

RNA from the FRR2161 type strain (the wild-type strain) was isolated from vegetative hyphal cells grown at 25°C for 2 days in liquid medium, from asexually developing cultures grown on solid medium at 25°C for 7 days, and from yeast cells grown at 37°C for 6 days in liquid medium. RNA was isolated from yeast cells derived either from LPS-activated J774 murine macrophages infected with wild-type conidia at 24 h postinfection or from yeast cells incubated in macrophage growth media for 24 h. Macrophages were infected as described in the "In vivo macrophage assay" section below. RNA was extracted using TRIzol reagent (Invitrogen) and an MP FastPrep-24 bead beater according to the manufacturer’s instructions. RNA was DNase treated (Promega) prior to RT-PCR analysis, and a synthesis control assay lacking cDNA was performed to ensure that no DNA contamination was present. Increasing numbers of cycles were used to ensure that the amplification was in the exponential phase, and the benA gene was used as a loading control. Expression of simA was determined by RT-PCR (Invitrogen Superscript III One-Step RT-PCR with platinum Taq) using primers simA-DD3 (5′-ATCCATCCCCCGTGAAGC-3′) and simA-DD4 (5′-GCCGACACGAAGTGATCC-3′). Band intensity was quantitated in Photoshop, and relative intensity values were calculated using the benA loading controls.

1 million PHA-activated PBMCs were coinfected in 300 μl medium containing equal amounts of wild type and frameshift viruses encoding a rev1-vpu fusion gene or not. After 6 h, cells were washed three times and cultured in 2 ml of supplemented RPMI containing 10 ng/ml IL-2. Supernatants were collected 10 d after infection and RT-PCR (SuperScript III One-Step RT-PCR with Platinum Taq; Invitrogen) was performed to amplify viral genomic RNA using primers flanking the rev1-vpu intergenic region. The PCR fragments were purified from agarose gels and sequenced to determine the outcome of the competition.

RNA was amplified simultaneously using a universal primer set adapted to the conserved 3′ and 5′ segment ends for full-length amplification of all influenza A viruses (Hoffmann et al., 2001 (link)) using Superscript III One-Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA, United States). The PCR conditions were as described in an article by King (King et al., 2020 (link)). After amplification, the samples were purified with AMPure XP Magnetic Beads (Beckman Coulter, Fullerton, CA, United States) in a × 0.65 sample volume to bead volume ratio. Quantification was conducted with the Qubit 4 Fluorometer (Thermo Fisher Scientific).
Following the manufacturer’s instructions, the Native Barcoding kit (cat. no. ONT EXP-NBD104) and the Ligation kit (cat. no. ONT SQK-LSK109) were used. After library preparation, the pooled samples were loaded onto a FLO-MIN106 R9.5 flow cell. An 8-h run was conducted with standard settings.