Ampure xp pcr beads

Tag- and wild type–specific amplicons from cells used in the experiments shown in Fig. 4 were generated from 200 ng gDNA using junction-specific primers (Table S4) by a two-step nested PCR with Velocity polymerase (Bioline). The first PCR reaction was performed for 15 cycles with 60°C annealing and 30 s elongation and then purified with AMPure XP PCR beads (Beckman Coulter). The second PCR was performed for 15 cycles for wild type–specific and for 21 cycles for tag-specific amplicons, respectively, using 60°C annealing and 30 s elongation. PCR products were size selected by gel electrophoresis on 2% agarose/TAE and gel extracted by column purification (Macherey-Nagel). Amplicons were paired-end sequenced with 500 cycles on a MiSeq system (Illumina) using the Amplicon-EZ (150–500 bp) service by Genewiz to acquire at minimum 13,123 reads per sample. Paired reads were merged and aligned to the respective expected amplicon references using CRISPResso (v2.0.29; Kleinstiver et al., 2019 (link)) with parameters “cleavage_offset,” 1; and “window_around_sgrna,” 0. Mutations were subsequently quantified using a custom R script excluding primer binding sites in the analysis.