Mus musculus
Protocols: 17 Cl-1 cells were grown on 100-mm dishes to 90% confluency and infected with MHV-A59 at a multiplicity of infection (MOI) of 10, or treated with tunicamycin for 6 hours at 2ug/mL. At indicated 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)]. The 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 and then 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. Due to the very large amounts of vRNA produced during infection, mock samples were processed separately from infected samples, at all stages of nucleic acid extraction and library construction, to avoid contamination. 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.
ENA-FIRST-PUBLIC
2020-11-09T04:04:33Z
ENA-LAST-UPDATE
2020-01-10T13:35:57Z
External Id
SAMEA6465087
INSDC center name
Division of Virology, Department of Pathology, University of Cambridge
INSDC first public
2020-11-09T04:04:33Z
INSDC last update
2020-01-10T13:35:57Z
INSDC status
public
Submitter Id
E-MTAB-8650:5hpi_mock_2
broker name
ArrayExpress
cell line
17cl1 (RRID:CVCL_VT75)
cell type
fibroblast
common name
house mouse
developmental stage
spontaneously immortalised cell line
disease
normal
genotype
BALB/c
infect
mock
sample name
E-MTAB-8650:5hpi_mock_2
scientific_name
Mus musculus