GSM8709722: ONT_PCR_plasmid_libraries_DXYB6C_2_sample_2; synthetic construct; OTHER
Plasmids were purified with Zymo MidiPrep kit. AAV samples were sequenced by Plasmidsaurus. See manuscript for details.  To generate libraries of barcoded insert by PCR from the AAV packaged DNA, we first extracted DNA by performing proteinase K treatment (3 uL crude AAV prep, 6 uL 10 mM Tris 8, 1 uL proteinase K [Thermo Scientific EO0491], 60 min at 50C, 5 min at 70C, then placed on ice), followed by phenol/chloroform extraction (add 190 uL 10 mM Tris 8, add 200 uL phenol-chloroform-isoamyl alcohol [Invitrogen 15593-031], vortex 30 s, spin at 16,000 g at room temperature for 5 min, take aqueous layer) and isopropanol precipitation (add 1 uL glycoblue, 50 uL NaOAc 3M, 250 uL isopropanol 100%, vortex, 45 min at -80C, 45 min at 21,000 g at 4C, 80% EtOH wash, air dry pellet, resuspend in 10 uL 10 mM Tris 8). 2 uL of the precipitated DNA was taken as template for PCR with primers oJBL949+oJBL950 (Kapa Robust Hot Start Ready mix [Roche], 60C annealing temperature, elongation time 2 min 30 s) and tracked by qPCR (SYBr green). PCR libraries were generated from all samples packaged with the PHP.eB serotype in standard (non-sparse) packaging conditions (i.e. samples 1 to 6, see Fig. S4f). Two separate reactions per sample (reaction 1: volume 20 uL, 16 cycles; reaction 2: volume 50 uL, 17 to 20 cycles) were pooled prior to submission. Of note, we tested the importance of adding a DNase treatment step by performing qPCR with primers targeting the insert between the AAV ITRs (oJBL905+oJBL906) vs. a sequence on the ampicillin cassette in the backbone (oJBL097+oJBL098) comparing with/without DNase treatment prior to proteinase K treatment but saw no difference (>30-fold lower backbone material relative to cargo), likely because of the benzonase treatment already having degraded the non-encapsidated DNA. PCR libraries from plasmids were prepared from 1 ng of template (with quantification possibly confounded by genomic DNA carryover), and reactions were stopped at 18 cycles (input starting material calibrated to still be in exponential phase before the inflection point). Plasmid-templated samples also included the parental p146 (which was pooled at 1/10-fold stoichiometry of other larger inserts to mitigate ONT size biases). In both plasmid- and AAV-templated conditions, PCR reactions were cleaned up with 0.4x Ampure clean, leaving predominantly >2 kb products, Fig. S5g (i.e., largely excluding p153×-short and p153×-mid samples). Two samples corresponding to pooled products (AAV pool sample 1, plasmid pool sample 2) were submitted to Plasmidsaurus for long-read sequencing (custom project ID: DXYB6C, target reads 1M per sample).  PCR amplicons were submitted to Plasmidsaurus for sequencing by Oxford Nanopore Technology.