shRNA knockdown

EB Erica M. Briggs
PM Paolo Mita
XS Xiaoji Sun
SH Susan Ha
NV Nikita Vasilyev
ZL Zev R. Leopold
EN Evgeny Nudler
JB Jef D. Boeke
SL Susan K. Logan
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DUSP1 and scramble shRNA were cloned into a pTRIPZ backbone. pTRIPZ plasmid and viral packaging plasmids were transfected into HEK 293 T cells with Lipofectamine Reagent (Thermo Fisher 18324012) (2 μg pMD2G, 3 μg psPAX2, and 5 μg pTRIPZ) and virus was collected and filtered after 48 h. Viral supernatant (4 mL) was supplemented with fresh media (2 mL) and Polybrene (8 μg/mL) and incubated with E006AA-hT cells for 4 h. After 48 h, cells were treated with puromycin (1 μg/mL). Once selected, shRNA expression was induced with 1 μg/mL doxycycline for 48 h.

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