Pacbio SEQ2 Short insert library: Arabidopsis 3 week seedlings
A. thaliana seeds were surface sterilized with 75% ethanol, plated on half strength Murashige and Skoog (MS) agar medium at 4°C in the dark for 3 days until uniform germination, and then were germinated on at 22°C in constant light for 10 days. The seedlings with about similar size were transferred to potting mix for growth in 22°C growth chambers with 16-h light/8-h dark cycles. The plant materials of whole seedlings were collected at day 21 after initiation of germination. Genomic DNA was extracted using a DNeasy Plant Mini kit (Qiagen) following manufacturers' instructions. The short-insert DNA SMRTbell library was performed according to the manufacturer's instructions by first shearing the sample to ~200bp using a Covaris LE220 Focused-ultrasonicator, followed by damage and end repair and ligation of single-stranded barcoded adaptors. Sequencing primer was then annealed to the SMRTbells and incubated with the template for 1 hour at 20 ºC using a ratio of 20:1. The Sequel 2.1 polymerase was then bound to the annealed template complex using a ratio of 30:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 °C. The binding complex was cleaned up, diluted to the final loading concentration, and held at 4 °C until diffusion-based loading on the Sequel II instrument. Diluted binding complex at concentration of 125-175pM was loaded on the 8M sequencing chip and 30 hour movies were recorded according to the manufacturer's guidelines.