Pippin prep machine

Two separate CRISPR primer (CRISPR1 and CRISPR2 locus) pairs were designed for two first rounds of PCR. Two microliters of template DNA entered a first round of PCR. The primer design incorporates a recognition sequence to allow a secondary nested PCR process. Samples were first purified with Ampure SPRI Beads before entering the second PCR performed to incorporate Illumina adapter sequences. Samples were purified using Ampure SPRI Beads before being quantified using Qubit and assessed for size distribution using the Fragment Analyzer (Agilent). Successfully generated amplicon libraries were taken forward and pooled in equimolar amounts, then size selected with a Pippin Prep machine (Sage Science) using a range of 180–600 bps. The quantity and quality of each pool was assessed by Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa on a Roche Light Cycler LC480II according to the manufacturer’s instructions. Template DNA was denatured according to the protocol described in the Illumina cBot User guide (Illumina Document #15006165 v05) and loaded at 12.5 pM concentration. Fragmented PhiX phage genome was added to the sequence library at 15% in order to increase the sequence complexity. The sequencing of each pool was carried out on one lane of an Illumina MiSeq, at 2×250 bp paired-end sequencing with v2 chemistry.