Truseq nano dna lt library preparation kit

For Illumina sequencing, the phenol/chloroform extraction protocol was used to extract DNA from 2 g of young leaves. An Illumina sequencing library for an insertion length of 250 bp was prepared using the TruSeq Nano DNA LT Library Preparation Kit (Illumina Inc., San Diego, CA, USA). DNA purity and size range were evaluated with Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). An Illumina sequencing library (PE) with an insertion length of 300–350 bp was constructed and sequenced using the Illumina HiSeq 2000 platform.
The DNA extracted from the young leaves was also used for the PacBio sequencing library construction. According to the manufacturer’s protocol (Pacific Biosciences, San Diego, CA, USA), 10 μg of Chinese flowering cabbage genomic DNA was used for a 30-kb template library preparation using the BluePippin Size Selection system (Sage Science, Waltham, MA, USA). The library was sequenced on the PacBio SEQUEL II platform.
The PacBio platform was used to generate long genomic reads for the construction of a reference genome for the Chinese flowering cabbage. After removing adaptor sequences, more than 113 Gb of subreads were obtained with a 219 times sequence coverage. The sequencing data were used for the following genome assembly operations.

For DNA extractions, 10–20 mg of silica-dried stem or leaf tissue was ground with a Qiagen TissueLyser II (Qiagen, Melbourne, VIC, Australia). DNA was extracted using the DNeasy 96 Plant Kit or DNeasyPlant Mini Kit (Qiagen) following the manufacturer’s protocol and eluted in 100 μl of TE buffer (Qiagen).
Sequencing libraries were constructed from 100 ng of total DNA using the TruSeq Nano DNA LT library preparation kit (Illumina, San Diego, CA, United States) for an insert size of 350 base pairs (bp), following the manufacturer’s protocol. Sequencing libraries were multiplexed 96 times and DNA sequencing with 125-bp paired-end reads was performed on an Illumina HiSeq 2500 platform at the Australian Genomic Research Facility (AGRF, Melbourne, VIC, Australia).

The PowerFecal DNA isolation kit (MO BIO Laboratories, Inc., San Diego, CA) was used to extract DNA per protocols established by the Human Microbiome Project.⁴⁷ Library construction and sequencing was carried out at the New York Genome center using the TruSeq Nano DNA LT Library Preparation Kit (Illumina, Inc., San Diego, CA), which generates 450 bp libraries and sequenced on the HiSeq 2500 System at 2 × 125 bp read length resulting in ~250 M reads.