Is it recommended to transduce all three lentiviral particles simultaneously and perform a triple antibiotics selection? Or would it be more prudent to transduce each particle sequentially and perform individual selection?
Are you using the SAM activation library? It's generally better to do it step-wise (as mentioned in the Joung et. al. Nat. Protocols paper) since doing a triple antibiotic selection might potentially have toxic effects on your cells and you are adding another layer of variability to the screen. I would suggest generating stable cell lines first and then using those to perform the screen.
Hope this helps!
I wasn't doing a screen but a gene specific activation. I had viral particles for dCas9, sgRNA for a specific gene, and MS2, and infected the cells with them simultaneously. As you have correctly pointed out, the triple antibiotics combination was really toxic, together with the variability of only a small population of cells taking up three viral particles, had wiped out all the cells relative to untransduced cells.
I will redo the transduction with only dCas9 and MS2, select for positive cells, before transducing the sgRNAs.