title: PacBio of Mus musculus: liver
design: Samples were collected, and high molecular weight genomic DNA was prepared by the CTAB method and followed by purification with Grandomics Genomic kit for regular sequencing, according to the standard operating procedure provided by the manufacturer. The DNA degradation and contamination of the extracted DNA was monitored on 1% agarose gels. DNA purity was then detected using NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific, USA), of which OD260/280 ranging from 1.8 to 2.0 and OD 260/230 is between 2.0-2.2. At last, DNA concentration was further measured by Qubit 4.0 Fluorometer (Invitrogen, USA).SMRTbell target size libraries were constructed for sequencing according to PacBios standard protocol (Pacific Biosciences, CA, USA) using 15kb preparation solutions. The main steps for library preparation are: (1) gDNA shearing, (2) DNA damage repairend repair and A-tailing, (3) ligation with hairpin adapters from the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences), (4) nuclease treatment of SMRTbell library with SMRTbell Enzyme Cleanup Kit, (5) size selection, and (6) binding to polymerase. Briefly, a total amount of 8 g DNA per sample was used for the DNA library preparations. The genomic DNA sample was sheared by g-TUBEs (Covaris, USA) according to the expected size of the fragments for the library. Single-strand overhangs were then removed, and DNA fragments were damage repaired, end repaired and A-tailing. Then the fragments ligated with the hairpin adaptor for PacBio sequencing. And the library was treated by nuclease with SMRTbell Enzyme Cleanup Kit and purified by AMPure PB Beads. Target fragments were screened by the PippinHT (Sage Science, USA). The SMRTbell library was then purified by AMPure PB beads, and Agilent 2100 Bioanalyzer (Agilent technologies, USA) was used to detect the size of library fragments. Sequencing was performed on a PacBio Sequel II instrument with Sequencing Primer V5 and Sequel II Binding Kit 2.2 in Grandomics.