GSM8524121: N2c_293T_Lipofectamine2000_Gpseudotyped_15cm-2; Lyssavirus rabies; OTHER
phenol-chloroform extraction to extract viral RNA genomes; gigaprep to extract plasmid DNA To quantify plasmid barcode library diversity, 50 ng of plasmid DNA was tagmented using seqWell's Tagify UMI reagent, in order to tag each barcode-containing plasmid DNA fragment with a UMI and partial Illumina Read1 adapter. Tagmented fragments were purified using a 1× left-sided size selection using SPRIselect beads according to the manufacturer's protocol. Sample indexing PCR was subsequently performed using custom sample index primers that bind to the BFP primer site sequence immediately proximal to the barcode sequence and to the TruSeq P5 PCR handle generated during Tn5 tagmentation (Primers 3 and 4, Key Resource Table). The library was sequenced using a target of 100 million reads. To quantify viral barcode library diversity, EnvA-pseudotyped barcoded RVdG was concentrated by ultracentrifugation as described above and RNA was isolated by phenol-chloroform extraction, the procedure for which is described as follows. The viral pellet was resuspended in 1 mL of Trizol (Invitrogen 15596026), and incubated at room temperature for five minutes to permit dissociation of rabies proteins from viral RNA. Subsequently, 200 μL of chloroform (Fisher 60047878) was added and vortexed for 30 seconds, followed by spin at 12000xg for 15 minutes at 4°C. 600 μL of the upper aqueous phase was collected and RNA was precipitated by mixing with 500 μL of isopropanol and 1 μL of Glycoblue (Invitrogen AM9515) and incubating for 10 minutes. Precipitated RNA was pelleted by centrifugation at 12000xg for 10 minutes at 4°C, washed twice with 80% ethanol, air dried for 2 minutes, and resuspended in 10 μL of nuclease-free water. Reverse transcription was performed on 1-5 μg of RNA using SuperScript IV (ThermoFisher 18090010) and 1 μL of 50 mM custom RT primer targeting a sequence distal to the barcode relative to the negative stranded viral genome, in order to generate cDNA containing barcodes from viral genome RNA but not from contaminant mRNA. RT primer also contained an overhanging sequence carrying an 8 bp randomer, which functions as a UMI to correct for PCR amplification bias after sequencing, and a binding site for Illumina sample index primer. The resulting cDNA (5 uL, or 25% of the RT reaction) was amplified using 10-12 cycles of PCR with KAPA HotStart PCR ReadyMix (Roche KK2601) and custom sample index primers, one of which is specific to an overhang introduced by the RT primer to facilitate viral genome-specific amplification. Each library was sequenced using at least 10 million reads.