ERC000011
ENA-CHECKLIST
2023-12-01T00:42:37Z
ENA-FIRST-PUBLIC
2023-12-01T00:42:37Z
ENA-LAST-UPDATE
SAMEA113449597
External Id
Division of Virology, Department of Pathology, University of Cambridge
INSDC center name
2023-12-01T00:42:37Z
INSDC first public
2023-12-01T00:42:37Z
INSDC last update
public
INSDC status
ERS15445037
SRA accession
E-MTAB-12967:PE243_Vero_REP2
Submitter Id
ArrayExpress
broker name
Vero
cell_line
fibroblast
cell_type
Protocols: Vero and U251 cells were grown on 100-mm dishes to 90% confluency and infected with ZIKV (PE243 or 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 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.
description
adult
developmental stage
normal
disease
wild type genotype
genotype
ZIKV PE243
infect
Chlorocebus sabaeus
organism
Chlorocebus sabaeus
organism
kidney
organism part
Chlorocebus sabaeus
scientific_name
PE243_Vero_REP2
title