Six weeks after subretinal injection, RPEs were isolated using an established protocol with minor modification.49 (link) In brief, after enucleation of the eye, anterior chamber, lens, and retina were removed, and the posterior eye cup was incubated with 0.05% trypsin-EDTA for 45 min at 37°C. Followed by gentle shaking of the eye cup to isolate the RPEs from the eye cup, cells pallets were collected for genomic DNA extraction.
Genomic DNA was extracted using either a DNeasy blood and tissue kit (QIAGEN, 69504) or a QIAamp DNA FFPE tissue kit (QIAGEN, 56404) according to manufacturer’s instructions, and the quantity and quality of gDNA were measured using a NanoDrop 2000c device (Thermo Fisher). PCR products were amplified from 100 ng of gDNA with forward and reverse primers using Phusion high-fidelity polymerase (NEB, M0530S) for 35 cycles (98°C for 10 s, 60°C for 30 s, and 72°C for 15 s) then purified with a MiniElute PCR purification kit (QIAGEN, 28004). The primer pairs used in this study are listed inTable S1 . Sanger sequencing was performed at the UC Davis DNA sequencing facility. Deep sequencing of 200-bp PCR products of VEGF-A target sites amplified from genomic DNA was conducted at the DNA-sequencing core facility at Massachusetts General Hospital (Boston, MA, USA).
Genomic DNA was extracted using either a DNeasy blood and tissue kit (QIAGEN, 69504) or a QIAamp DNA FFPE tissue kit (QIAGEN, 56404) according to manufacturer’s instructions, and the quantity and quality of gDNA were measured using a NanoDrop 2000c device (Thermo Fisher). PCR products were amplified from 100 ng of gDNA with forward and reverse primers using Phusion high-fidelity polymerase (NEB, M0530S) for 35 cycles (98°C for 10 s, 60°C for 30 s, and 72°C for 15 s) then purified with a MiniElute PCR purification kit (QIAGEN, 28004). The primer pairs used in this study are listed in