GSM9113028: Viral library 12 well rescue; Lyssavirus rabies; OTHER
For viral libraries, viral genomic RNA was extracted using the Quick DNA/RNA Viral Kit (Zymo Research, D7020) and eluted into 15 μl of RNase-free water. Purified viral barcode RNA was then reverse transcribed using Superscript IV reverse transcriptase (Thermo Fisher Scientific, 18090050) at 50 °C for 10 minutes with 1 μl of 2 μM UMI_P5_Truseq primer (Supplementary Table 1), before heat inactivation at 80 °C for 10 minutes. RNA was subsequently digested with RNase H (New England Biolabs, M0297S) at 37 °C for 20 minutes. Barcoded library sequencing. The diversity of the barcoded plasmid and viral libraries was quantified using a unique molecular identifier (UMI) tagging approach.  The primer used for reverse transcription (UMI_P5_Truseq, Supplementary Table 1) contained an overhang of 22 bp P5 Truseq for nested adaptor PCR, a 12 bp random UMI sequence and 20 bp of rabies homologous sequence for binding upstream of the barcode. The same UMI-P5-Truseq primer was used to produce UMI-tagged amplicons from the barcoded plasmid libraries in a two-cycle limited PCR reaction. Barcoded plasmid (5 ng) was added to 16 μl Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, M0494S) with 1.25 μl each of UMI_P5_Truseq and P7_universal_reverse primers (Supplementary Table 1) and PCR thermocycled with the following conditions: 98 °C - 3 mins, two cycles of 98 °C - 30 s, 67 °C - 20 s, 72 °C - 30 s, followed by 72 °C - 2 mins and 12 °C hold. The resultant cDNA from the viral samples or amplicons from barcoded plasmids was size-selected and purified using KAPA Pure Beads (Roche, KK8000) in a 1:1 ratio, eluting in 25 μl of RNase-free water. To determine the optimal cycle number for library amplification, the amplification curve inflection point was determined for each sample by qPCR. 2.5 μl of each sample was added to a master mix of 1 μl 2.5X Thiazole Green (Biotium, 40086), 1 μl 2.5X ROX reference dye (Thermo Fisher Scientific, 12223-012), 12.5 μl Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs, M0494S), 1.25 μl 10 μM IDT_i7_1 primer (Supplementary Table 1) for plasmid amplicons or P7_universal_reverse for viral single-stranded cDNA, 1.25 μl 10 μM IDT_i5_1 primer and 5.5 μl of molecular grade water. The following thermocycler conditions were used: 98 °C - 3 mins, followed by 40 cycles of 98 °C - 10 s, 68 °C - 20 s, 72 °C - 30 s. Subsequently, the remaining 22.5 μl of each sample was added to a mix of 25 μl Q5 Hot Start High-Fidelity 2X Master Mix, 2.5 μl 10 μM IDT_i7_1-10 primer for plasmid amplicons/P7_universal_reverse for viral single-stranded cDNA, and 2.5 μl 10 μM IDT_i5_1-10 primer. The IDT_i5_X and IDT_i7_X primers each contain 8 bp index sequences derived from the rhAmpSeq index primer set (Integrated DNA Technologies) for dual-index demultiplexing alongside the Illumina P5 and P7 adapter sequences, respectively. Thermocycling conditions for this Illumina adaptor PCR were: 98 °C - 3 mins followed by cycles of 98 °C - 10 s, 68 °C - 20 s, 72 °C - 30 s, then 72 °C - 2 mins. The number of cycles was equal to the qPCR inflection point, less 3 cycles to account for the 10X (∼23) increase in target concentration. For viral amplicons, the P7-indexed adaptor sequences were added in a final PCR step. The UMI-tagged viral amplicons were first gel-purified with a Wizard SV Gel and PCR Clean-Up kit (Promega, A9281) and eluted with 20 μl water. Then, the same PCR mix was used as described above for the barcoded plasmid index PCR, adding the IDT_i7_X primer and the matching IDT_i5_X for each sample. Thermocycling conditions were likewise the same as the Illumina adaptor PCR, but with a fixed number of 3 cycles. The indexed viral and plasmid amplicons were then gel-purified with a Wizard SV Gel and PCR Clean-Up kit (Promega, A9281) and eluted with 25 μl water. The concentration and size distribution of these amplicon libraries were quantified with a High Sensitivity D1000 ScreenTape kit (Agilent, 5067-5584) on an Agilent 2200 TapeStation system. Samples passing QC were sequenced on an Illumina NovaSeq 6000/NovaSeq X using Illumina TruSeq primers for paired-end 100 bp sequencing at a depth of 10-50 million reads per sample.