A template-driven synthesis platform enables scalable kilobase-long sequence variants up to 10kb. NBThighlight
EcoRI. The two products were purified using SPRI beads, ligated using T4 DNA ligase , and incubated at 16 °C overnight. A pooled plasmid was transformed into competent NEB Turbo cells and grown for 10 h at 37 °C. Individual colonies were selected for colony PCR with Oxford Nanopore sequencing barcoded primers. Indexed amplicons were pooled in an equimolar fashion and sequenced on the Oxford Nanopore platform to identify mutation sites as well as barcode sequences.
AAV virus was produced using AAVpro Helper Free System , with minor adjustments. In brief, a 150 mm cell culture dish was inoculated with 6.0 × 10293T cells in DMEM culture medium supplemented with 1× GlutaMAX, 1× Pen–Strep antibiotic, and 5% FBS according to standard cell culture protocols. The 293T cells were split into ten 150 mm cell culture dishes for the experiment when cells were approximately 90% confluent.
To evaluate the packaging efficiency of the variants, barcode regions of variants for input plasmids and virus particles were quantitatively amplified and sequenced on the NextSeq platform. In brief, 1 μl purified AAV2 particles and input plasmid pools underwent 12-cycle PCR amplification using AAV_bcRead primer pairs and cleaning up with SPRI beads to generate amplicons of barcode regions.
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