mock
Protocols: BSR cells were grown on 90-mm dishes to 80% confluency and infected with TMEV at a multiplicity of infection (MOI) of 3, or mock-infected. At 10 hpi, cells were rinsed with 5 ml of warm PBS, flash frozen in liquid nitrogen, 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 25-G needle. Cell lysates were clarified by centrifugation at 15,000 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. 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 35-65nt. Library amplicons were constructed adapters based on the TruSeq small RNA adapters, with an additional seven random nucleotides at the 5′-end of the 3′-adapter.
ENA first public
2021-07-31
ENA last update
2021-07-31
External Id
SAMEA7192775
INSDC center alias
Division of Virology, Department of Pathology, University of Cambridge
INSDC center name
Division of Virology, Department of Pathology, University of Cambridge
INSDC first public
2021-07-31T00:10:54Z
INSDC last update
2021-07-31T00:10:54Z
INSDC status
public
Submitter Id
E-MTAB-9437:mock
broker name
ArrayExpress
cell line
BSR
common name
golden hamster
developmental stage
neonate
disease
normal
infect
mock
organism part
kidney
sample name
E-MTAB-9437:mock