One shot stbl3 chemically competent e coli cells

cDNA synthesis was performed from total RNA extracted from t-hESC and primed with oligo(dT), using SuperScript III First Strand Synthesis System (Thermo Fisher Scientific). Wild type SAE2 and UBC9 ORF were amplified by RT-PCR using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) and subcloned into pUC19 vector using InFusion HD Cloning Kit (Takara Bio USA). Both, SAE2 and UBC9 ORFs were then PCR amplified and subcloned into pLV-mCherry plasmid (Addgene #36084). All PCR steps described above have been performed using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific). All subcloning steps have been performed in OneShot Stbl3 Chemically Competent E. coli cells (Thermo Fisher Scientific). Lentiviral expression plasmids were co-transfected with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) as previously described(Benoit et al., 2017 (link)). Medium was collected, centrifuged to remove cells and debris, filtered through a 0.45um filter and then ultracentrifuged at 20K x g for 2hr at 4°. Concentrated lentivirus was re-suspended in 0.5ml of PBS per 75cm² flask of 293-FT cells worth of supernatant. Individual lentivirus preps were titrated on the desired cell line to be transduced in order to optimize expression but minimize transduction induced cell death.