Maxwell rsc 48

Genomic DNA used for sequencing was extracted using a combination of bead-beating (FastPrep-24, MP Biomedicals, Irvine, CA) and magnetic-bead purification (Maxwell RSC 48, Promega, Madison, WI). First, isolates from Sabouraud Dextrose agar plates were mixed with silica beads (Lysing Matrix C, MP Biomedical) and then mechanically sheared with 2 cycles at 6.0 m/s for 30 s with a 5 min pause between (FastPrep-24, MP Biomedical). Genomic DNA was extracted using PureFood Pathogen Kit (Promega) on a Maxwell RSC 48 (Promega) using manufacturer’s protocol. Genomic DNA was library prepped using DNA Prep Kit (Illumina, San Diego, CA) using manufacturer’s recommended protocol using a STARlet automated liquid handler (Hamilton Company, Reno, NV). Paired-end sequencing (2×151) was performed using Illumina’s MiniSeq and NovaSeq 6,000 to a minimum depth of 35x average coverage.

Viral loads were measured by Virology Services (WNPRC). vRNA was isolated from plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit on the Maxwell 48RSC instrument (Promega, Madison WI). vRNA was then quantified using a highly sensitive qRT-PCR assay based on the one developed by Cline et al. (48 (link)). RNA was reverse transcribed and amplified using TaqMan Fast Virus 1-Step Master Mix (Invitrogen) on the LightCycler 480 or LC96 instrument (Roche, Indianapolis, IN) and quantified by interpolation onto a standard curve made up of serial ten-fold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from the p239gag_Lifson plasmid, kindly provided by Dr. Jeffrey Lifson, (NCI/Leidos). The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5′- GTCTGCGTCATCTGGTGCATTC-3′, reverse primer: 5′-CACTAGCTGTCTCTGCACTATGTGTTTTG-3′ and probe: 5′-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-BHQ1-3′. The reactions cycled with the following conditions: 50°C for 5 min, 95°C for 20 s followed by 50 cycles of 95°C for 15 s and 62°C for 1 min. The limit of detection of this assay is 100 copies/mL.

Viral loads were measured by Virology Services (WNPRC). vRNA was isolated from plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit on the Maxwell 48RSC instrument (Promega, Madison WI). vRNA was then quantified using a highly sensitive qRT-PCR assay based on the one developed by Cline et al. [104 (link)]. RNA was reverse transcribed and amplified using TaqMan Fast Virus 1-Step Master Mix qRT-PCR Master Mix (Invitrogen) on the LightCycler 480 or LC96 instrument (Roche, Indianapolis, IN) and quantified by interpolation onto a standard curve made up of serial ten-fold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from the p239gag_Lifson plasmid, kindly provided by Dr. Jeffrey Lifson, (NCI/Leidos). The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5′- GTCTGCGTCATCTGGTGCATTC-3′, reverse primer: 5′-CACTAGCTGTCTCTGCACTATGTGTTTTG-3′ and probe: 5′-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-BHQ1-3′. The reactions cycled with the following conditions: 50°C for 5 min, 95°C for 20 s followed by 50 cycles of 95°C for 15 s and 62°C for 1 min. The limit of detection of this assay is 100 copies/mL.