PacBio Smrt cell sequencing of Anolis distichus, smart cell 2
We contracted the University of Delaware Sequencing and Genotyping Center to perform Pacific Biosciences high-fidelity long-read sequencing. High molecular weight (HMW) genomic DNA was extracted using the MagAttract HMW DNA Kit (Qiagen Inc., Venlo, Netherlands) according to the manufacturers instructions. The presence of HMW DNA was confirmed using a FemtoPulse (Agilent, Santa Clara, CA).  10 $\mu$g of DNA was converted into SMRTBell templates using the HiFi Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA). Unligated adapters and damaged DNA fragments were removed using exonuclease treatment and small fragments (<5kbp) were removed using BluePippin (Sage Science Inc., Beverly, MA). Size distribution verification and library quantification was performed using a FemtoPulse (Advanced Analytical Technologies Inc., Ankeny, IA) with 1 $\mu$L of library as input. Before sequencing, primers were annealed and DNA polymerase was bound to the libraries using the Sequel II Binding Kit 2.0 (Pacific Biosciences, Menlo Park, CA). Libraries were sequenced on the Sequel IIe with 8M SMRT cells using chemistry 3.0, with settings of four hour pre-extensions and 30 hour movie time.