CRISPR/Cas9-mediated stable knockout cells and reconstitution.

Cells were transfected with pLentiCRISPRv2 plasmids containing the gRNAs targeting indicated genes (Figure 7-figure supplement 1) using polyethyleneimine. After 24 hours, cells were selected with puromycin for another 48 hours and seeded into 96-well plates with 1 cell in each well. After 2 weeks, the single clones were selected from 96-well plates for further validation by Western blotting and DNA sequencing (Figure 7-figure supplement 1). Each knockout clone was generated with single gRNA.

pLenti CMV Neo DEST expression vectors containing empty or indicated gene constructor were packed into lenti-virus with the packaging vector psPAX2, the envelope vector pMD2.G, and polyethyleneimine. Cells were infected with the indicated virus and selected with puromycin for ~5 days, after 24 hours. The pooled cells were confirmed with Western blotting to validate the expression of genes and used in further experiments.