Nebnext ultra 2 q5 pcr master mix

Genomic DNA was extracted from ~5 × 10⁶ LCv2 transduced cells per condition using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The AAVS1 gene locus was amplified by PCR with the NEBNext Ultra II Q5 PCR Master Mix (NEB) at the following conditions: 34 cycles (98°C-10s, 67°C-10s, 72°C-120s) (see Supplemental Table 2 for primer sequences). All PCR reactions were purified using the AxyPrep Mag PCR Clean-up (Axygen Inc, Corning, NY) according to the manufacturer’s instructions. PCR performed on this pooled genomic DNA yielded a mix of amplicon products, which were then sequenced to yield >65,000 reads per condition. CRISPR amplicon sequencing was performed by the CCIB DNA Core Facility at Massachusetts General Hospital (Cambridge, MA). The gene editing efficiency was calculated using the CRISPResso2 alignment tool (www.crispresso.pinellolab.partners.org/submission)^{10 (link)}. Sequencing reads were deposited in the Sequence Read Archive (PRJNA812897). To check if the common sequencing primers could amplify the LCv2-7SK-Blast sgRNA cassette, 1μg of genomic DNA was amplified by PCR with the NEBNext Ultra II Q5 PCR Master Mix (NEB) at the following conditions: 24 cycles (98°C-10s, 63°C-10s, 72°C-120s) (see Supplemental Table 2 for primer sequences). PCR products were run on a 2% agarose gel.

The DeepHF design tool^{15 (link)} was used to predict the optimal sgRNA activity for both sgEBV1 and sgEBV4. The RefSeq EBV annotation NC_007605.1 was used for the design of both sgRNAs. The probability of sgEBV4 generating a frameshift in the beta-barrel region of the DNA-binding and dimerization domain of EBNA1 was predicted with inDephi^{16 (link)}. All sgRNAs (IDT) were cloned into lentiCRISPRv2 (Addgene #52961, we call this vector LCv2-U6-Puro) and lentiCRISPRv2 blast (Addgene #98293), and/or the new lentiCRISPR-7SK-Blast (Addgene #183620) using the Esp3I (BsmBI) cloning site (New England Biolabs, Ipswich, MA) as described^{1 (link)}. sgRNA sequences are listed in Supplemental Table 1. To check if the common sequencing primers could amplify LCv2-7SK-Blast, 20pg of plasmid DNA was amplified by PCR with the NEBNext Ultra II Q5 PCR Master Mix (NEB) at the following conditions: 34 cycles (98°C-10s, 67°C-10s, 72°C-120s) (see Supplemental Table 2 for primer sequences). PCR products were run on a 2% agarose gel.