Illumina Seq of Saccharum hybrid 25OFF2: CRISPR Mutant WT
212 bp amplicons of the 25OFF2 target region were generated using Q5 polymerase (reaction conditions: 98C for 10 secs, 29 cycles of amplification at 98C for 10 s, 69C for 30 s, 72C for 20 s, and a final extension at 72C for 2 min. Reactions were then purified using a GeneJET PCR Purification Kit (K0701) (Thermo Fisher Scientific). CRISPR amplicon sequencing was performed at the CCIB DNA Core Facility at Massachusetts General Hospital (Cambridge, MA). Adapters with unique barcodes were ligated onto each sample during the library construction. Libraries were pooled into equimolar concentrations for multiplexed sequencing on the Illumina MiSeq platform (Illumina, San Diego, CA).