title: PacBio: Agrobacterium_rosae_rho-6.1
design: SMRTbell template libraries were prepared according to the instructions from Pacific Biosciences (Menlo Park, CA, USA), following the Procedure & Checklist  Greater Than 10 kb Template Preparation document. Briefly, for preparation of 15 kb libraries, 8 g genomic DNA was sheared using g-tubes from Covaris (Woburn, MA, USA) according to the manufacturers instructions. DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosciences. Reactions were carried out according to the instructions of the manufacturer. BluePippin Size-Selection to greater than 4 kb was performed according to the manufacturers instructions (Sage Science, Beverly, MA, USA). Conditions for annealing of the sequencing primers and binding of polymerase to purified SMRTbell template were assessed with the Calculator in RS Remote (Pacific Biosciences). Single-molecule real-time (SMRT) sequencing was carried out on the PacBio RSII (PacificBiosciences) taking one 240-minutes movie on one SMRT cell per sample using the P6 Chemistry.