PACBIO of Gallus gallus: BAC-clone containing rRNA
Library preparation and sequencing were performed according to the Shared protocol-20kb Template Preparation Using BluePippin Size Selection system (15 kb size Cutoff). At each step, the DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). DNA purity was tested using the Nanodrop (Thermofisher), while size distribution and degradation were assessed using the Fragment analyzer (AATI) HighSensitivity Large Fragment 50kb Analysis Kit. Purification steps were performed using 0.45X AMPure PB beads (PacBio). High quality BAC DNA were extracted using the Nucleobond Xtra Midi Plus kit (Macherey Nagel) following the manufacturers instructions using 100 mL of LB media with Chloramphenicol selective marker (12.5 g/mL). DNA and END damage repair step was performed using SMRTBell template Prep Kit 1.0 (PacBio). Then blunt hairpin adapters were ligated to the library. The library was treated with an exonuclease cocktail to digest unligated DNA fragments. A size selection step using a 10kb cutoff was performed on the BluePippin Size Selection system (Sage Science) with the 0.75% agarose cassettes, Marker S1 high Pass 15-20 kb. Conditioned Sequencing Primer V2 was annealed to the size-selected SMRTbell. The annealed library was then bound to the P6-C4 polymerase using a ratio of polymerase to SMRTbell at 10:1. Then, after the magnetic bead-loading step (OCPW), the SMRTbell library was sequenced on 1 SMRTcell on RSII instrument at 0.25 nM with a 360 min movie.