Dsdna 915 reagent kit

Whole genome sequencing was performed using DNBSEQ-G400 (MGI Tech Co., Ltd.). Libraries were prepared using IMGIEasy FS DNA Library Prep Set (MGI Tech Co., Ltd.) and fragments were validated using Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies). Sequences were de novo assembled using SPAdes assembler version 3.15.5 42 (link). Genome annotation was performed using DFAST.

The concentration of DNA solution was quantified using SynergyLX (BioTek, United States) and QuantiFluor dsDNA System (Promega, United States). DNA was amplified using the 2-step tailed PCR to target the V3-V4 regions of bacterial 16S rRNA. 1st PCR was performed with the 1st-341f_MIX (5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-CCTACGGGNGGCWGCAG-3′) and the 1st 805r_MIX (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-NNNNN-GACTACHVGGGTATCTAATCC-3′) primers. Subsequently, 2nd PCR was performed with the 2ndF (5′-AATGATACGGCGACCACCGAGATCTACAC-Index2-ACACTCTTTCCCTACACGACGC-3′) and the 2ndR (5′- CAAGCAGAAGACGGCATACGAGAT-Index1-GTGACTGGAGTTCAGACGTGTG-3′) primers. Each PCR reaction was carried out using Ex Taq HS (Takara Bio Co., Shiga, Japan). V3-V4 PCR products were purified using AMPure magnetic beads (Beckman Coulter, United States) and quantified with Synergy H1 (BioTek, United States) and QuantiFluor dsDNA System (Promega, United States). Next, the PCR products were pooled to construct the sequencing library and the quality of the library was confirmed using Fragment Analyzer and dsDNA915 Reagent Kit (Advanced Analytical Technologies, United States). Sequencing was performed using the MiSeq Reagent Kit v3 (Illumina, United States) under the condition of 2 × 300 bp.

Flowers at the bloom stage were sampled and immediately frozen in liquid nitrogen. Three biological replicates for the WT and line #170 were used for RNA sequencing (RNA-Seq), and each biological replicate contained at least five blooming flowers. Total RNA was extracted with TRIzol reagent according to the manufacturer’s protocol. RNA quality was measured using a total RNA Quantus Fluorometer and QuantiFluor RNA system (Promega, USA). RNA quantity was assessed using a Qsep100 DNA Fragment Analyzer and RNA R1 Cartridge (BiOptic, Taiwan). The libraries were prepared using an MGIEasy RNA Directional Library Prep Set (MGI, China) according to the manufacturer’s instructions. The concentration of the libraries was measured by Qubit and a dsDNA HS Assay Kit (Thermo Fisher Scientific, USA), and the quantity was checked using Fragment Analyzer and a dsDNA 915 Reagent Kit (Advanced Analytical Technologies, UK). Paired-end reads (150 bp) were generated on the DNBSEQ-G400 at Bioengineering Lab. Co. (Kanagawa, Japan).

Total RNA was isolated from cells using the PureLink RNA Mini Kit (12183018A) according to the manufacturer’s instructions. RNA concentration was analyzed by Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States), and the purity was assessed using the Qsep100 DNA Fragment Analyzer and RNA R1 Cartridge (BiOptic, New Taipei City, Taiwan). Subsequently, total RNA was converted to cDNA and used for Illumina sequencing library preparation based on the KAPA Stranded mRNA-Seq Kit protocols (KAPA Biosystems, Wilmington, MA, United States). DNA fragments were then subjected to adapter ligation, where dsDNA adapters with 3’-dTMP overhangs were ligated to A-tailed library insert fragments by FastGene Adapter Kit (NIPPON Genetics, Bunkyo, Tokyo, Japan). The purified cDNA library products were evaluated using Qubit and dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States), followed by quality assessment using the Fragment Analyzer and dsDNA 915 Reagent Kit (Advanced Analytical Technologies, Ankeny, IA, United States) and finally by sequencing (2 × 75 bp) on NextSeq 500 (Illumina, San Diego, CA, United States).