Chromium genome library kit gel bead kit v2

For library preparation and sequencing, one nanogram of high quality, high molecular weight DNA was used for the 10x Genomics linked-read library preparation using the Chromium Genome Library Kit & Gel Bead Kit v2, the Chromium Genome Chip Kit v2, and the Chromium i7 Multiplex Kit according to the manufacturer’s instructions (10x Genomics, Pleasanton, USA). The library quality was assessed using Bioanalyzer DNA High Sensitivity DNA Assay (Agilent) and the concentration was tested with Qubit^® dsDNA BR Assay Kits (Thermo Fisher Scientific). The 10x Genomics library was sequenced on the Illumina HiSeq X Ten (150 bp paired-end reads). For RNA, we constructed cDNA libraries according to the MGIEasy RNA Library Prep set protocol. The libraries were sequenced with the MGISEQ-2000RS machine.

High-molecular-weight DNA was isolated from leaf material of seedlings of ‘Haruna Nijo’²² and size selected for a molecule size of 40 kb or higher. The 440-bp paired-end (PE) libraries were prepared with the Hyper Kapa Library Preparation kit (Kapa Biosystems) with no polymerase chain reaction amplification. The 8- to 10-kb mate-pair (MP) libraries were constructed with the Nextera Mate Pair library Sample Prep kit (Illumina, San Diego, CA, USA) followed by the TruSeq DNA Sample Prep kit. The 10X libraries were constructed with the Chromium Genome Library Kit & Gel Bead Kit v2 (10X Genomics). Sequencing was performed following Sato et al.²¹ In brief, the 440-bp PE libraries were sequenced for 251 cycles using a NovaSeq 6000 system (Illumina). The 10X and MP libraries were sequenced for 151 cycles from each end of the fragments on the NovaSeq 6000 system. All libraries were prepared and sequenced at the University of Illinois Roy J. Carver Biotechnology Center (Urbana, IL, USA). In situ Hi-C libraries were prepared as described by Padmarasu et al.²³ (link) Sequencing data generated from each of the libraries are listed in Supplementary Table S1. The Hi-C data were used to prepare chromosome-scale assemblies using the TRITEX pipeline,¹⁹ (link) which was also used for the contig assembly and scaffolding with the PE, MP, and 10X data (Supplementary Table S1).

For whole-genome sequencing, 1.25 ng of high integrity, high molecular weight DNA was used for the 10x Genomics linked-read library preparation using the Chromium Genome Library Kit & Gel Bead Kit v2, the Chromium Genome Chip Kit v2 and the Chromium i7 Multiplex Kit according to the manufacturer’s instructions (10x Genomics, Pleasanton, CA, USA). The 10x Genomics library was sequenced on the Illumina HiSeq X Ten (150 bp paired-end reads). To obtain short-read RNA sequences, the sequencing library was prepared according to the method reported in Pootakham et al. (2021b (link)). A total of 200 ng of poly(A) mRNA was used to construct a library using the MGIEasy RNA Library Prep Kit V3.0 (MGI Tech, Shenzhen, China). The library was subsequently sequenced using the MGISEQ-2000RS Sequencing Flow Cell V3.0 and the MGISEQ-2000RS (MGI Tech, Shenzhen, China).