Qseq100 dna analyzer

DNA was extracted from 100 mg fresh young leaf tissue using the CWBIO Plant Genomic DNA Kit (CoWin Biosciences, Beijing, China), according to the manufacturer’s protocol. DNA was quantified using a Nanodrop 2000 spectrophotometer based on absorbance of 260 nm (Thermo Scientific, Waltham, MA, USA). DNA concentrations were adjusted to 50 ng/μl and subsequently used for RAD library preparation. DNA was digested by the EcoRI restriction enzyme (New England BioLabs, NEB), then ligated to a unique barcode adapter. Samples were pooled together and randomly sheared ultrasonically. The average length of sheared fragments was confined to around 400 bp using Qseq100 DNA Analyzer (Bioptic Inc.). The AMPure XP Beads PCR Purification kit (Beckman Coulter, Inc.) was applied to purify the sheared DNA fragments. The fragment ends were repaired with the Quick Blunting kit (NEB). A 3′-dA overhang was added using the dA-tailing module contained in the kits (NEB), and then ligated to a common adapter. The collected fragments were enriched by PCR amplification and purified using the AMPure XP Beads PCR Purification kit (Beckman Coulter, Inc.). Each sample was normalized to 10 nM for sequencing using a Hi-seq X Ten (Illumina) at ORI-GENE (Beijing, China). The RAD sequences were submitted to the NCBI Sequence Read Archive with BioProject ID: PRJNA503672.

Following the Illumina sequencing platform manufacturer’s instructions, first, the first and the second strand were synthesized. Next, the cDNA was purified, ends were repaired, ligated to the adapter, followed by library enrichment. The cDNA quality and concentration were evaluated using Qseq100 DNA Analyzer (Bioptic Inc, China). Finally, the Illumina HiSeq 2000 platform was used to sequence the cDNA library.