Qubit rna 2.0 fluorometer

Clarified infectious cell culture supernatant was filtered using 0.22-μm filters (Merck Millipore Co., MA, USA) to remove possible cellular residues. RNA was extracted using QIAamp viral RNA minikit (Qiagen, Hilden Germany) following the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Qubit RNA 2.0 fluorometer using the Qubit RNA HS assay kit (Invitrogen, USA). Libraries for sequencing were prepared using TruSeq stranded mRNA kit (Illumina, USA), following the manufacturer’s protocol with the modification to exclude the poly(A)-containing mRNA purification steps. Reverse transcription was done using Superscript III reverse transcriptase (Invitrogen, USA) and random hexanucleotide primers (Invitrogen, USA). This was followed by second-strand synthesis using DNA polymerase I and RNase H, provided with the library preparation kit. Purification was performed using AMPure XP beads (Beckman Coulter, USA) after which the purified double-strand cDNA fragments were end repaired by adding a single A’ nucleotides to the 3′ end of the blunt fragments. Ligation of the adapters was performed, and the products purified and enriched by PCR to create the final library. Libraries were normalized, pooled, and sequenced using the Illumina platform.

The clarified supernatant of the 5 individual bulk pools was passed through 0.22-μm filters to remove excess host “contaminants” and any bacteria while concentrating the viral particles. In preparing the 5 samples, one extraction blank and two positive samples were included as controls. The two positive-control samples were dengue virus type 2 isolates that had been amplified in Vero cells. RNA extraction was performed using a QIAamp viral RNA minikit (Qiagen, Germany) following the manufacturer’s recommended protocol. RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Qubit RNA 2.0 fluorometer using the Qubit RNA HS assay kit (Invitrogen, USA). The RNA was then prepared for Illumina library preparation.