Genejet plant genomic dna purification mini kit

The lines selected for sequencing were grown in a greenhouse and leaf tissue was collected 3 weeks after planting. For each genotype, a bulked sample of 12 plants were collected and leaf tissue was lyophilized and ground. Genomic DNA was extracted using the GeneJet Plant Genomic DNA purification mini kit (Thermo Scientific, Boston, MA, USA) and 150-bp DNA fragments were sequenced with the NextSeq Sequencing instrument (Illumina, San Diego, CA). Adapters were removed from the raw Fastq files using Trimmomatic v0.36 (Bolger et al., 2014 (link)), and sequencing reads were mapped to the soybean genome Wm82.a2.v1 (https://data.jgi.doe.gov) with Bowtie2 v2.3.3.1 (Langmead and Salzberg, 2012 (link)). SNP and indel calls were performed with the GATK HaplotypeCaller software (McKenna et al., 2010 (link)) and variants were annotated with SNPeff version 4.3t (Cingolani et al., 2012 (link)). Variant visualization in the Chr 20 QTL region was performed with the Integrative Genomics Viewer (IGV - v2.9.5) (Robinson et al., 2011 (link)).

Genomic DNA from leaf tissue of Kuras was extracted using Gene Jet Plant Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Waltham USA), and used to amplify two fragments of 508 and 684 bp of the GBSS gene (Supplementary Fig. S1). 250 ng of DNA was used as template in a PCR with 0.5 µmol of primers (Supplementary Table S1) StGBSSExf and StGBSSExr, and primers StGBSSExon3f and StGBSSExon3r (Sigma-Aldrich, St. Louis, USA), Phusion HF polymerase, dNTP, and HF buffer (Thermo Fisher Scientific, Waltham, USA) according to the supplier’s instructions. The PCR was as follows: 98 °C for 1 min, 30 cycles at 98 °C for 10 s, 64 °C for 10 s, 72 °C for 15 s and final extension at 72 °C for 10 min.
The PCR product was ligated to pJET1.2/blunt using a CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, USA) following transformation to One Shot^® TOP10 Chemically Competent Escherichia coli (Invitrogen, Carlsbad, USA). After overnight incubation at 37 °C on LB plates containing 100 μg/mL ampicillin, plasmids were purified from 12 randomly picked colonies and insert sequenced (GATC Biotech, Konstanz, Germany).

Genomic DNA was extracted from Desiree leaf tissue using the Gene Jet Plant Genomic DNA purification Mini Kit (Thermo Fisher Scientific, Waltham, MA) and sequenced through the Illumina TruSeq PCR-free library preparation (average fragment size 350 bp) and Illumina HiSeqX (PE 2 × 150 bp) (National Genomics Infrastructure, Stockholm, Sweden). Targets were selected in regions of exon 5 in Sbe1 (GenBank accession no. NW_006238958.1:c2098376-2090439) and in exon 3 in Sbe2 (GenBank accession no. NW_006238947.1: c2592132-2611729) using CRISPR RGEN Tools (http://www.rgenome.net/cas-designer) and a previously available guide design tool (http://crispr.mit.edu/). Double sgRNA targets were selected for each gene and named BE1T3 and BE1T4 as well as BE2T3 and BE2T4 (Fig. S1). All sgRNAs were designed to target allelic homologous regions, except for BE2T4 which had a mismatch at the first bp prior PAM in one of the four alleles.