RiboSeq of African green monkey (Vero) cells infected at a multiplicity of infection (MOI) of 3 with the Asian/American Zika virus (ZIKV) strain, PE243
RiboSeq of African green monkey (Vero) cells infected at a multiplicity of infection (MOI) of 3 with the Asian/American Zika virus (ZIKV) strain, PE243
Vero cells were grown on 100-mm dishes to 90% confluency and infected with the American/Asian Zika virus (ZIKV)strain, PE243, 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 spontaneously immortalised cell line recovered and stored in liquid nitrogen. RNase I was added to the lysates for 45 minutes at room temperature, following which ribosomes and ribosome-protected fragments of RNA were purified by centrifugation through a sucrose cushion. RNA was extracted from the purified ribosomes 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.