We measured genome-wide off-target editing using CHANGE-seq described by Lazzarotto et al.5 with minor modifications. The genomic DNA purification steps were carried out using the NEB Monarch Genomic DNA Purification Kit. An agarose gel was used to visualize the tagmentation of the human genomic DNA with the transposase. For the PCR step after cleavage and USER enzyme treatment (step 25 of the supplemental information ), NEB 2X Q5 Master Mix was used in place of 2X Kapa HiFi HotStart Ready Mix. In place of the MiSeq protocol described in the supplemental information , cleaved genomic DNA barcoded and amplified via PCR for illumina sequencing was sent for NGS Amplicon-EZ sequencing by Azenta.
Hifi hotstart ready mix
Manufactured by New England Biolabs
The HiFi HotStart Ready Mix is a high-fidelity, hot-start PCR master mix designed for robust and reliable amplification of DNA. It contains optimized buffer, MgCl2, dNTPs, and a proprietary hot-start DNA polymerase.
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2 protocols using hifi hotstart ready mix
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Genome-wide off-target editing analysis
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Genome-wide Off-target Editing Analysis
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We measured genome-wide off-target editing using CHANGE-seq described by Lazzarotto et al.5 with minor modifications. The genomic DNA (human male/female mixed, Promega #G3041) purification steps were carried out using the NEB Monarch Genomic DNA Purification Kit. An agarose gel was used to visualize the tagmentation of the human genomic DNA with the transposase. For the PCR step after cleavage and USER enzyme treatment (step 25 of the supplemental information), NEB 2X Q5 Master Mix was used in place of 2X Kapa HiFi HotStart Ready Mix. In place of the MiSeq protocol described in the supplemental information, cleaved genomic DNA barcoded and amplified via PCR for illumina sequencing was sent for NGS Amplicon-EZ sequencing by Azenta.
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