The virus-related methods have been previously described (Xie and Hon, 2019 (link)). Briefly, the Ientivirus packaging plasmids MD2.G and psPAX2 (Addgene ID 12259 and 12260) were co-transfected with the carrier plasmid to HEK293T cells with linear polyethylenimine (PEI, Polysciences). Supernatant was collected 72 h after transfection and filtered with a 0.45μm filter. The virus was further purified by using Lenti-X Ientivirus concentrator (Clontech). For virus titration, we performed infection of K562 cells with serially diluted virus, and measured the survival rate of the cells after the antibiotic selection. For the single-cell enhancer screen, we performed infection of K562 cells in 24-well. We tried different multiplicity of infection (MOIs) in order to maximize the number of sgRNAs we could test per cell. We found a non-linear relationship between the MOI of virus and the actual number of sgRNA detected (Table S2 ). By using MOI = 2000, we were able to detect about 10 sgRNAs per cell.
Lenti x ientivirus concentrator
Manufactured by Takara Bio
The Lenti-X Lentivirus Concentrator is a laboratory equipment designed to concentrate lentiviral particles. It utilizes a proprietary membrane-based technology to efficiently concentrate lentiviral vectors from cell culture supernatants or other liquid samples.
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2 protocols using lenti x ientivirus concentrator
1
Single-Cell Enhancer Screening Using Lentivirus
2
Single-Cell Enhancer Screening Using Lentivirus
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The virus-related methods have been previously described (Xie and Hon, 2019 (link)). Briefly, the Ientivirus packaging plasmids MD2.G and psPAX2 (Addgene ID 12259 and 12260) were co-transfected with the carrier plasmid to HEK293T cells with linear polyethylenimine (PEI, Polysciences). Supernatant was collected 72 h after transfection and filtered with a 0.45μm filter. The virus was further purified by using Lenti-X Ientivirus concentrator (Clontech). For virus titration, we performed infection of K562 cells with serially diluted virus, and measured the survival rate of the cells after the antibiotic selection. For the single-cell enhancer screen, we performed infection of K562 cells in 24-well. We tried different multiplicity of infection (MOIs) in order to maximize the number of sgRNAs we could test per cell. We found a non-linear relationship between the MOI of virus and the actual number of sgRNA detected (Table S2 ). By using MOI = 2000, we were able to detect about 10 sgRNAs per cell.
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