Short-read Illumina sequencing of Neofelis nebulosa: whole blood
Genomic DNA was isolated from a ~400 uL aliquot of whole blood from each sample using the Mag-Bind Blood and Tissue Kit (Omega Bio-Tek Inc., Georgia, USA). The concentration and integrity of the gDNA was checked with the Picogreen method using Victor X2 fluorometry (Life Technologies, Carlsbad, CA) and a genomic DNA ScreenTape assay using an Agilent 4200 Tapestation (Agilent Technologies, Santa Clara, CA), respectively. The gDNA was then sheared with a Covaris S220 Ultrasonicator (Covaris, Woburn, MA) to enrich fragments of 350 base pairs (bp), which were used to prepare a genomic library using the TruSeq DNA PCR-free library kit (Illumina, San Diego, CA). The template size distribution of the PCR enriched fragments was verified using an Agilent 4200 Tapestation and the libraries were quantified with a Lightcycler qPCR assay (Roche Life Science, St. Louis, MO). The libraries were paired-end sequenced (2 x 150 bp) to a depth of 20x in an S4 flow cell on an Illumina NovaSeq 6000 instrument (Illumina, San Diego, CA).