Purelink rna mini kit columns

Competitor viruses were competed with an MHV-ExoN(-) virus harboring 10 silent mutations in the probe-binding region within nsp2. Subconfluent DBT-9 monolayers in 24-well plates were coinfected at a total MOI of 0.01 PFU/cell with competitor and reference viruses at a 1:1 ratio and passaged 4 times. For each passage, supernatants were harvested at 24 h. RNA was extracted from 100 μl of supernatant using 900 μl of TRIzol reagent and PureLink RNA minikit columns (Thermo Scientific, Waltham, MA), and 150 μl of supernatant was used to infect fresh cells in a 24-well plate (total MOI estimated at 1 PFU/cell). The proportion of each virus was determined by real-time RT-qPCR from the infection supernatant using two TaqMan probes with different fluorescent dyes in separate reactions. Competitor viruses were detected with the same probe used in specific infectivity analyses (14 (link)). Reference viruses were detected by a probe targeting the same region but with 10 silent mutations (5′-TCCGAACTACTGCAACCCCAAGTG-3′) and labeled with 5′ quasar 670 and 3′ black hole quencher 2 (BHQ-2) (Biosearch Technologies, Petaluma, CA). RNA copy numbers were calculated by reference to an RNA standard generated by in vitro transcription of the corresponding MHV A fragment, and relative RNA abundances were calculated as ratios of competitor genomes to reference genomes.

RNA was isolated from cells with TRIzol reagent followed by purification over PureLink RNA mini kit columns (Invitrogen). RNA-seq was performed using a poly-A library construction protocol with strand-specific cDNA synthesis and 8 cycles of PCR. Purification of poly-A+ mRNA was performed by flow-through total RNA using MultiMACS 96 Separation Unit. For library construction, 96-well Plate-based Strand-specific cDNA Synthesis using Maxima H Minus Strand Specific 96-well was used for Illumina HiSeq 2500 Sequencing.