) targeting the N-terminus of TRIM28 was cloned into the sgRNA-Cas9 vector pX458 . The repair template and the sgRNA-Cas9 vector were transfected into V6.5 mESCs and iPSCs by nucleofection using Amaxa 4D Nucleofector X Unit according to the manufacturer’s instructions. To screen for positive integrations, the transfected cells were sorted for mRuby2 fluorescent protein expression with flow cytometry. The sorted cells were seeded as single cells and expanded for a few days.
was mixed with mESC medium and incubated for the time indicated; medium was changed daily for fresh dTAG-13.. For IF, dTAG-13- or DMSO- treated cells were fixed in 4% paraformaldehyde for 10 min at RT and stored in PBS at 4 °C. All buffers and antibodies were diluted in RNase-free PBS . Cells were permeabilized with 0.5% Triton X-100 for 10 min at RT, followed by three consecutive 5 min PBS washes.
NatureGenet 😬 zombies embryoniccells 🧟♀️
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