organism: Chlorocebus sabaeus
description: Protocols: Vero cells were grown on 100-mm dishes to 90% confluency and infected with ZIKV (Dak84) at a multiplicity of infection (MOI) of 3. At 24 h p.i., cells were rinsed with 5 ml of ice-cold PBS, flash frozen in a dry ice/ethanol bath and lysed with 400 μl of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 μg/ml cycloheximide and 25 U/ml TURBO DNase (Life Technologies)]. Cells were scraped extensively to ensure lysis, collected and triturated ten times with a 26-G needle. Cell lysates were clarified by centrifugation at 13,000 g for 20 min at 4°C. The supernatants were recovered and stored in liquid nitrogen. RNA was extracted from the lysates by proteinase K digestion (enzyme from NEB, reaction at 42 degrees for 30 minutes) followed by hot acidic phenol: chloroform extraction and ethanol precipitation. The methodologies employed were based on the original protocols of Ingolia and colleagues (Ingolia et al., 2009; Ingolia et al., 2012), except library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina smallRNA v2 to allow multiplexing. Ribosomal RNA was removed using Ribo-Zero Gold rRNA removal kit (Illumina), and remaining RNA was purified by 15% denaturing urea gel purification to select fragments of 25-35nt, and library amplicons were constructed.