Amplicon sequencing of Maize protoplasts: WT rep 5
Maize B104 plants were used to generate protoplasts. The protoplasts were transfected with a plasmid containing the Cas9 coding sequence as a negative control. Two days after transfection, DNA was extracted from the protoplasts using the Edwards protocol (Edwards et al, 1991). DNA samples were quantified using Qubit. 50 amplicons for 40 genes were amplified in a multiplex PCR per sample, while adding sample-specific indices (Floodlight Genomics, USA). Illumina sequencing adapters were ligated using the KAPA Hyper Prep PCR-free Kit according to manufacturer directions (Kapa Biosystems, USA) and libraries were paired-end sequenced (PE-150) on an Illumina HiSeqX instrument (Admera Health, USA). All read data were demultiplexed, barcodes and adapter sequences were removed, and reads were trimmed and merged.