Chlorocebus sabaeus
Protocols: Vero cells were grown on 100-mm dishes to 90% confluency and infected with different ZIKV mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) 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 the remaining RNA was fragmented by alkaline hydrolysis (45mM NaHCO3, 6mM Na2CO3, 1mM EDTA; 95 degrees, 15 minutes). Fragments of 25-35nt were selected by 15% denaturing urea gel purification, and library amplicons were constructed.
ENA-FIRST-PUBLIC
2023-12-01T00:42:39Z
ENA-LAST-UPDATE
2023-12-01T00:42:39Z
External Id
SAMEA113537019
INSDC center name
Division of Virology, Department of Pathology, University of Cambridge
INSDC first public
2023-12-01T00:42:39Z
INSDC last update
2023-12-01T00:42:39Z
INSDC status
public
Submitter Id
E-MTAB-12968:American WT_RNA
broker name
ArrayExpress
cell_line
Vero
cell_type
fibroblast
disease
normal
genotype
wild type genotype
infect
ZIKV American WT
organism part
kidney
sample name
E-MTAB-12968:American WT_RNA
scientific_name
Chlorocebus sabaeus