MISSION® shRNA ZEB1 Lentiviral Transduction Particles

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Infect at MOI of 3:1 in CMRL 1066 medium containing 8 μg/ml polybrene for overnight. After 4 days select cells with 1 μg/ml puromycin for 3 days.

Protocol tips
Infect at MOI of 3:1 in CMRL 1066 medium containing 8 μg/ml polybrene for overnight. After 4 days select cells with 1 μg/ml puromycin for 3 days.

Publication protocol

RIP-Cre/ER and pTrip–loxP-Stop-loxP-eGFP lentiviruses7 were used for lineage tracing. Virus production, cell infection, and tamoxifen treatment were previously described7. eGFP-labeled cells were sorted using a FACS Aria sorter as described6,7. shRNA cloned in pLKO.1-PURO lentiviral vectors (TRCN-017563 and -017566 for ZEB1, TRCN-013528 for ZEB2, TRCN-355694 and -010772 for SOX2, TRCN-017988 and -017991 for SOX6, and scrambled control) were obtained from Sigma-Aldrich. For ZEB inhibition, cells were infected at MOI of 3:1 in CMRL 1066 medium containing 8 μg/ml polybrene (Sigma-Aldrich) overnight. Four days following infection the cells were selected with 1 μg/ml puromycin for 3 days. For miR-200c retrovirus production, the pre-mmu-miR-200c miR-VEC-Blasticidin vector42 was co-transfected into human embryonic kidney 293T cells with the Ampo-helper packaging plasmid. The medium was replaced 6 h post-transfection, and the virus was harvested 24 h later and used fresh. 1 × 106 cells were plated in 14-cm culture dishes in growth medium for 24 h. Cells were infected at MOI of 3:1 in medium containing 8 μg/ml polybrene for 6 h. The infection was repeated two more times in the following two days. Selection was initiated 2–3 days later with 4 μg/ml blasticidin for 7 days. Following selection (a total of 10 days from the first infection), the cells were harvested for analysis

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for MISSION® shRNA ZEB1 Lentiviral Transduction Particles below.

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