SAMN02639795
External Id
UCLA
INSDC center name
2014-03-07T00:00:00Z
INSDC first public
2021-09-22T10:00:48.698Z
INSDC last update
SRS557324
INSDC secondary accession
live
INSDC status
Pathogen.cl
NCBI submission model
Pathogen.cl.1.0
NCBI submission package
SRS557324
SRA accession
Hangfei Qi
collected by
2010
collection date
A plasmid carrying the genotype 2A hepatitis C viral genome was subjected to in vitro bacteriophage Mu transposon mutagenesis, followed by incubation on plates containing kanamycin and ampicillin to select for bacteria transformed with transposon-inserted plasmids. Pooled plasmids were isolated and digested with Not I enzyme to remove the transposon fragment, followed by a self-ligation step, which left fifteen nucleotides randomly inserted across the virus genome. This library DNA was used for in vitro transcription, then transfected into Huh 7.5.1 cells en masse. Total cellular and viral RNA was extracted at 96 hours (96h) post-electroporation, then reverse transcribed into cDNA. The cDNA then served as template for PCR to amplify thirteen overlapping fragments covering the entire virus genome. These amplicons were randomly fragmented, insertion sites enriched, and pooled PCR products were sequenced by Illumina sequencing.
description
2a
genotype
USA: Los Angeles
geo loc name
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
host
Hepatitis C
http://www.ebi.ac.uk/efo/EFO_0003047
host disease
Huh 7.5.1 cell line
host_description
Laboratory cell culture
isolation source
34.10305555 N 118.35777777 W
lat lon
Hepacivirus hominis
organism
15-nt insertion library pool 1 passaged in cell culture for 96h
title