Extended Data Fig. 10: General workflow of establishing INSPECT-tagged cells by positive and negative selection.
From: Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs
Cells are transfected or electroporated for delivery of the CRISPR-Cas9–i53 plasmid as well as the INSPECT donor plasmid. Cells are then cultured until puromycin resistance is established. After up to one week of puromycin selection (concentration depends on cell line), cells are transfected or electroporated with an expression plasmid coding for CAG-promoter-driven mesostable Flp recombinase (P2S, L33S, Y108N, S294P) to remove the selection cassette (PuroR and HSV-TK) from the genome. Before monoclonalization, cells were counter-selected using ganciclovir (2-10 µM). The medium must be changed regularly to avoid the accumulation of toxic ganciclovir triphosphate. Cells are finally genotyped for determination of zygosity, and sequence integrity of the splice sites is verified via Sanger sequencing.
