CRISPR 128 genomic zebrafish loci – Canonical – Mutant pool2
Amplicon quality and concentration were determined by estimating the A260/A280 and A260/A230 ratios by nanodrop. Amplicon integrity and size were confirmed by running an Agilent Bioanalyzer gel. 100 ng of PCR amplicons (1-2kb) were sheared to 120-150bp using a Covaris E210 instrument. Following shearing, a SPRI bead cleanup was performed using Ampure XP SPRI (Beckman Coulter Genomics). A shearing QC was then performed on sheared products using the DNA 1000 Bioanalyzer chip to confirm the target size. Sheared amplicons were then end-repaired and A-tailed and adapters were ligated. Indexed libraries that meet appropriate cut-offs were quantified by both qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with a LabChip GX. Samples with a yield of ≥0.5 ng/ul were used for sequencing. Sample concentrations were normalized to 2 nM and loaded onto Illumina version 3 flow cells at a concentration that yields 170-200 million passing filter clusters per lane. Samples were then sequenced using 75 bp paired-end reads on an Illumina HiSeq 2000 according to Illumina protocols. The 6 bp index is read during an additional sequencing read that automatically follows the completion of read 1. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time. Primary analysis and sample de-multiplexing were performed using Illumina's CASAVA 1.8.2 software suite.
Yale University
crispr_rules_can_mutant2_AG00839