Pacbio SEQ2 Short insert library: Chlamydomonas reinhardtii CC-125 mt+
The short-insert DNA SMRTbell library was performed according to the manufacturers 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 C 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 manufacturers guidelines.