library (ORFeome Collaboration): DF/HCC DNA Resource Core. (Boston, MA), GE ... Wing Cheung (Medical University of South Carolina, Charleston,. SC).
Cloning the open reading frame expression library constructs The Invitrogen Gateway system (Thermo Fisher Scientific, Waltham, MA) was used to clone cDNA of the respective genes. Briefly, an entry vector containing the cDNA of interest was obtained from Addgene (Cambridge, MA) or distributors of the Human ORFEOME library (ORFeome Collaboration): DF/HCC DNA Resource Core (Boston, MA), GE Healthcare: Dharmacon RNAi and Gene Expression (Lafayette, CO), Arizona State University: DNASU Plasmid Repository (Tempe, AZ), and GeneCopoeia (Rockville, MD). The LR clonase enzyme was used to clone Gateway entry ORFs (pENTR223 backbone, pDONR223 backbone, pDONR221 backbone, or pShuttle backbone) into Gateway compatible lentiviral destination backbones (pLenti6.3/V5-DEST backbone (Thermo Fisher Scientific), pLX304 backbone (gift from David Root, Addgene plasmid #25890), pLEX_307 backbone (gift from David Root, Addgene plasmid #41392), and pLenti CMV HygroDest (w117-1) (gift from Eric Campeau, Addgene plasmid #17454)) encoding selection markers for either blasticidin, puromycin, or hygromycin, respectively. Reactions were stopped using proteinase K solution, promptly transformed into chemically competent One Shot Stbl3 cells (Thermo Fisher Scientific) or DH5α cells. Colonies were selected, propagated, stored, and plasmids were isolated. To confirm insert presence, plasmids were enzymatically digested with either the restriction enzyme BsrG1 or double digested with equal parts of Af1II and XhoI and reactions were stopped by the addition of 6X Bromophenol Blue then separated using ethidium bromide gel electrophoresis. Expression inserts were sequence verified by Eurofins MWG Operon (Louisville, KY) using flanking universal primers. The pLX304KAT6A plasmid was constructed and gifted from the laboratory of Dr. Hui Wing Cheung (Medical University of South Carolina, Charleston, SC).
