title: GSM9425752: PacBio_AAV_p152; synthetic construct; OTHER
library_construction_protocol: Plasmids were purified with Zymo MidiPrep kit. AAV samples were sequenced by Plasmidsaurus (ONT) or locally on a PacBio Vega. See manuscript for details on cloning of scQer-AAV libraries and other BC1-BC2 libraries used in the manuscript. AAV preps were submitted to Plasmidsaurus for direct sequencing (which involves an annealing step, end-repair and adapter ligation) with Oxford Nanopore. To generate libraries of barcoded insert by PCR from the AAV packaged DNA, we first extracted DNA by performing proteinase K treatment (3 μL crude AAV prep, 6 μL 10 mM Tris 8, 1 μL proteinase K [Thermo Scientific EO0491], 60 min at 50C, 5 min at 70C, then placed on ice), followed by phenol/chloroform extraction (add 190 μL 10 mM Tris 8, add 200 μLphenol-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 μL glycoblue, 50 μLNaOAc 3M, 250 μLisopropanol 100%, vortex, 45 min at -80C, 45 min at 21,000 g at 4C, 80% EtOH wash, air dry pellet, resuspend in 10 μL 10 mM Tris 8). 2 μL of the precipitated DNA was taken as template for PCR. Amplicons for the oBC-CRE-mBC association in scQer library were generated by PCR.