Generation of mouse haploid somatic cells by small molecules for genome-wide genetic screening
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE101795 | GEO | 2017/09/01
SECONDARY ACCESSION(S): PRJNA395545
REPOSITORIES: GEO
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Project description:U2OS cells were co-transfected with PiggyBac (PB) transposon plasmid pPB-SB-CMV-puro-SD3 and the transposase p-hyPBASE. The modified PiggyBac (PB) transposon, which has a constitutively active CMV promoter which can stimulate or disrupt expression of neighboring genes, depending on insertional orientation. For each library, between 10e7-10e8 cells were transfected, cultured with the addition of 3 µg/ml puromycin for one week to select cells that had incorporated the transposon, and then used for screens of resistance to alpha-toxin-induced cell death with minimal further expansion. Mutagenized cells were treated twice with alpha-toxin (500 ng/ml) for 48h to ensure the selection of only highly resistant cells. Genomic DNA (gDNA) was isolated from 5-10 X 10e6 cells and transposon insertion sites were mapped using high throughput sequencing.
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