Genomic DNA of Hunteria zeylanica
For the 48 newly sequenced plastomes, total genomic DNA was extracted using previously described methods (Livshultz et al., 2007), or using a FastPrep 120 homogenizer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), with FastDNA kit (MP Biomedicals, LLC, Santa Ana, California, USA). For some samples, 5 mg of dried leaf tissue was homogenized in 300  l of Tissue Lysis Buffer (Promega Corporation, Madison, Wisconsin, USA) and 5  l RNase A (10 mg/ml) using a FASTPrep 24 5G (MP Biomedicals) for 4-5 cycles of 40 m/s for 20 s, and then incubated at room temperature for 10 min. Then 30  l each of Lysis Buffer (Promega) and Proteinase K (Promega) were added and reactions incubated at 56 C for 20 min. Samples were then centrifuged at 14,500 x g for 2 min. The supernatant was transferred to Maxwell 16 LEV RNA extraction cartridges (Promega) for paramagnetic particle DNA extraction using a Maxwell 16 (Promega). DNA was visualized via agarose gel electrophoresis and quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Up to 1  g of extracted DNA was fragmented to approximately 300 - 400 bp by sonication in a Bioruptor Pico (Diagenode Inc., Denville, New Jersey, USA) for 8 cycles of 15 s on/90 s off or Bioruptor (Diagenode) for 5-15 cycles of 30 s on/30 s off at low power. Illumina libraries were prepared in one-half volume reactions (except for full-volume elution following enrichment) with NEBNext Ultra II library preparation kits (New England Biolabs Inc., Ipswich, Massachusetts, USA) with single index NextFlex HT adapters (Bioo Scientific, Austin, Texas, USA) and 8 cycles of polymerase chain reaction (PCR) for library enrichment. Bead-based size selection targeted inserts of 300-400 bp. For Skytanthus acutus Meyen, the library was prepared using a NEB adapter. For Metastelma northropiae Schltr., the library was prepared in a one-half volume reaction using the Illumina TruSeq Nano kit and an Illumina adapter (Illumina Inc., San Diego, California, USA) following the manufacturer s protocol. Library concentration was determined using a Qubit and final library size distribution was assessed with agarose gel electrophoresis. Libraries were pooled in equimolar ratio and 150 bp paired end reads were sequenced on a NextSeq 500 (Illumina) at the Oklahoma State University Bioinformatics Core or 75 bp paired end reads were sequenced on a MiSeq (Illumina) at Cornell University Institute of Biotechnology Genomics Facility.
SUB3375159
HUNZEY67cS12.01