Nextra xt index kit

For HMA, PCR on genomic DNA was performed over 35 cycles using KOD FX Neo (TOYOBO), and PCR products were analyzed using a microchip electrophoresis system (DNA-500 reagent kit and MCE-202 MultiNA; Shimadzu) according to Shigeta et al. (2016) (link). An amplicon-sequencing library was prepared based on the Illumina ‘16S Metagenomic Sequencing Library Preparation’. For the first round of PCR, the target regions containing sgRNA targeting sites were amplified from individual genomic DNA of uninjected control embryos (n=5), tyr (n=10), oca2 (n=5), and trβ (n=8) crispants using a KAPA HiFi HS ReadyMix (NIPPON Genetics, Tokyo, Japan) with primer sets containing barcode and overhang adaptor sequences. Equal quantities of all PCR products were pooled and purified using a QIAquick PCR Purification Kit (Qiagen). The second round of PCR was performed to construct a sequence library using a Nextra XT index kit (Illumina, CA, USA). The final library was purified and sequenced on the Illumina MiSeq. Library construction and sequencing were performed by Macrogen Japan and Hokkaido System Science. All primers are listed in Table S2.

The phagemid DNA from the SPINK2 library was analyzed using a HiSeq system (Illumina Inc.). The HiSeq library for DNA sequencing was prepared using a Nextra XT index kit (Illumina Inc.) following the protocol provided by the manufacturer. To amplify DNA for NGS, the PCR reaction was performed using the primers forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGATCCGCAGTTTGGTCTGTTTAGC-3′ and reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCCACCTTCACGAATTTTCATG-3′, and Pfu Turbo DNA polymerase (Agilent Technologies). Sequencing data were obtained from TAKARA BIO INC.

NGS was used to genotype the Pvtb1 mutant plants generated from micropropagation of primary mutants and BC1 Pvtb1 mutant progeny. To amplify the target fragments of each Pvtb1 gene, gene-specific primers were designed for Pvtb1a and Pvtb1b respectively. The amplicon size for Pvtb1a is 250 bp, while that of Pvtb1b is 288 bp, both are sufficiently long to cover the two target sequences. The Illumina overhang adapter sequences were added to the gene-specific primers (Table S6). A second round of PCR was used to add the dual indices with the Nextra XT Index Kit (Illumina, San Diego, CA, USA). The quality of these sequencing libraries was determined by the Qubit^® 2.0 Fluorometer. The 150-cycle HiSeq sequencing was done for all the amplicon libraries at the DNA Facility at Iowa State University (ISU). For each library, at least 5,000 reads were generated to determine the sequence of the respective amplicons. The results were analyzed by CRISPR-DAV pipeline (Wang et al., 2017 (link)).