ERC000011
ENA-CHECKLIST
2023-12-01T00:42:38Z
ENA-FIRST-PUBLIC
2023-12-01T00:42:38Z
ENA-LAST-UPDATE
SAMEA113537031
External Id
Division of Virology, Department of Pathology, University of Cambridge
INSDC center name
2023-12-01T00:42:38Z
INSDC first public
2023-12-01T00:42:38Z
INSDC last update
public
INSDC status
ERS15533456
SRA accession
E-MTAB-12970:Dak84_Ribo_U251_REP1
Submitter Id
ArrayExpress
broker name
U-251MG
cell_line
astrocytoid
cell_type
human
common name
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. 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.
description
adult
developmental stage
glioblastoma
disease
wild type genotype
genotype
ZIKV Dak84
infect
Homo sapiens
organism
Homo sapiens
organism
parietal lobe
organism part
Homo sapiens
scientific_name
Dak84_Ribo_U251_REP1
title