amplified gRNA from P23H Nrl-Cas9 retina
At P30, mice were sacrificed with CO2 and enucleated. Individual retinas were isolated immediately and lysed with homemade retinal lysis buffer containing 50 mM Tris, 1 mM CaCl2, 0.1% Tween-20 and 10 units / ml proteinase K (NEW ENGLAND Biolabs) overnight at 56C. Samples were centrifuged at 14,000 rpm at RT to remove tissue debris, then DNA was precipitated with 2-propanol, purified with 70% ethanol and dissolved with ddH2O. For DNA processing, sequencing, and downstream analysis, each individual retina served as an independent sample. Genomic DNA samples with low purity (260 nm/280 nm < 1.6) were excluded.  gRNA was amplified for sequencing from isolated DNA by nested PCR. For PCR1, all DNA from each sample was amplified within 50ul reactions each containing 50ug DNA using Q5 High-Fidelity DNA Polymerase (NEW ENGLAND Biolabs), an equimolar mix of phased Forward primers (ACGACGCTCTTCCGATCTTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTGCTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTAGCTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTCAACTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTTGCACCTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTACGCAACTTGTGGAAAGGACGAAACACCG, ACGACGCTCTTCCGATCTGAAGACCCTTGTGGAAAGGACGAAACACCG). and Reverse Primer (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTACTATTCTTTCCCCTGCACTGT) with the following conditions (50ul reaction; 98C 30s, 8 cycles [98C 10s, 55C 30s, 72C 30s], 10 cycles [98C 10s, 72C 60s], 72C 5min, 4C). Products were purified using SeraPure DNA isolation beads (Sera-mag SpeedBeads [Cytiva] prepared according to (Rohland and Reich 2012); 1:1 reaction:bead ratio, 2 washes with 80% Ethanol, and eluted in 25ul ddH2O). Eluted DNA from each experimental group was pooled from all PCR1 reactions and mixed before 2.5ul further amplified using Q5 High-Fidelity DNA Polymerase (NEW ENGLAND Biolabs) and barcoded primers with Illumina sequencing adaptors (F- AATGATACGGCGACCACCGAGATCTACAC[10bp-index]ACACTCTTTCCCTACACGACGCTCTTCCGATCT; R- CAAGCAGAAGACGGCATACGAGAT[10bp-index]GTGACTGGAGTTCAGACGTG) with the following conditions (50ul reaction; 98C 30s, 8 cycles [98C 10s, 66C 30s, 72C 30s], 5 cycles [98C 10s, 72C 60s], 72C 5min, 4C). Products were purified using SeraPure DNA isolation beads (1:1 reaction:bead ratio, 2 washes with 80% Ethanol, and eluted in 30ul ddH2O. All products were verified for size using a Bioanalyzer High Sensitivity DNA ChIP and DNA quantified by Qubit before submission for sequencing at the Washington University in St. Louis Genome Technology Access Center and the McDonnell Genome Institute. All libraries were sequenced using the Illumina NovaSeq X with 150bp paired end reads.