Fugene hd transfection reagent

The Brunello library (Addgene #73179) (Doench et al., 2016 (link)) was amplified in Endura Electrocompetent cells (Lucigen) and sequenced to confirm maintenance of sgRNA representation. Virus was generated by transfecting HEK293T cells growing in 10 cm dishes with 5 μg Brunello library plasmid, 3 μg psPAX2 (Addgene #12260), and 2 μg pMD2.G (Addgene #12259) using 30 μL FuGENE HD transfection reagent. Supernatant was collected at 48 hours and 72 hours post-transfection and stored at −80°C.

GC-C sequences were downloaded from GenBank based on whole genome sequences from each species tested. Each C-terminally HA tagged GC-C variant was synthesized via gene synthesis (Life Technologies). GC-C was then cloned into the lentiviral transfer vector pUltra (Addgene #24129) between the XbaI and BamHI restriction sites by Gibson Assembly, in frame with GFP and the T2A linker sequence. To generate lentiviral particles, 10 cm dishes were seeded with 3 × 10⁶ 293T cells 24 hours prior to transfection. Cells were then transfected with 7.6 μg pUltra-GC-C, 7.6 μg psPAX2 packaging plasmid (Addgene # 12260), and 3.8 μg pMD2.G envelope plasmid (Addgene # 12259) with 56 μl FuGene HD transfection reagent according to the manufacturer’s specifications. Media was replaced 24 hours post-transfection and replaced with 10 mL media. Viral supernatants were collected 48 hours-post transfection and passed through a 0.4 μm followed by overnight incubation with 1X PEG-IT solution (System Biosciences) at 4°C. Precipitated viral particles were centrifuged at 1500xg for 30 min at 4°C and resuspended in PBS at a final volume of 500 μl before storage at −80°C.