longamp-seq of hG6pc Cas9 treated mus musculus: adult liver
To further characterize the editing outcomes in GSDIa mice, we analyzed amplicons spanning the mG6pc gRNA target site using the approach recently described by Giorgi et al (50). For this method, we isolated genomic DNA from mouse livers and performed two cycles of PCR amplification with primers spaced approximately 750bp from the gRNA target site and which contained an 18bp unique molecular identifier flanked by a 24bp synthetic priming site (PCR1). Forward Primer: CAAGCAGAAGACGGCATACGAGATNNNYRNNNYRNNNYRNNNCACTGTTGGCTGCCCAAAC; Reverse Primer: AATGATACGGCGACCACCGAGATCNNNYRNNNYRNNNYRNNNGCCTGGCACATAGTAGCTGA. Dual-UMI tagged amplicons were then amplified by an additional 25 rounds of PCR using the synthetic priming sites (PCR2), followed by an additional 10 rounds of PCR using the synthetic priming sites with barcoded primers (PCR3) to facilitate multiplexing samples. All PCR reactions were performed with LongAmp Hot Start Taq DNA Polymerase (New England Biolabs, Cat# M0534L) according to the manufacturers recommendations with a 65 C annealing temperature and 18-minute extension time. After PCR1, excess UMI containing oligos were degraded by treatment with Exonuclease I (New England Biolabs, Cat# M0568L). After exonuclease treatment, and again after PCR2 and PCR3, amplicons were purified by SPRIselect beads (Beckman Coulter, Cat# B23317) using a 0.5X bead ratio. After the final purification, the HiFi library was prepared from 1g of pooled, barcoded amplicons and sequenced on a PacBio Revio instrument.