ZIKV Unpassaged Replicate 3
To enrich for encapsidated virus and to remove nucleic acids, 120 L of viral supernatant was mixed with 15 L of 250 units/mL of Benzonase (Millipore Sigma E1014-5KU) diluted in 10x Benzonase Buffer (200 mM Tris-Cl [pH 7.5], 100 mM NaCl, 20 mM MgCl2) and 15 L of 250 units/mL of RNAse A (Millipore Sigma 10109142001). Samples were then incubated at 37C for three hours. RNA was extracted from the samples using the Zymo Quick-RNA Viral Kit (R1035). After extraction, we purified the samples using a 0.8x bead selection with the sparQ PureMag magnetic beads (95196-005) to remove small RNA fragments. First-strand cDNA synthesis was performed using random nonamers and the Maxima H- Reverse Transcriptase kit (EP0751). We synthesized second-strand cDNA using the Q5 High-Fidelity 2X Master Mix (M0492S). NGS Libraries were then produced using the sparQ DNA Frag & Library Prep Kit from Quantabio (Cat. 95194-024).