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SI1.1. Tissue and scat sampling and DNA extraction
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Scat samples collected in the field were immediately stored separately in labelled re-sealable
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plastic bags, placed into portable coolers, and transferred to a -20°C freezer for storage until
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DNA extraction. Scat DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN)
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according to manufacturer’s protocol with some modifications described below.
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Each scat sample was sub-sampled using sterile forceps at three surface locations (ends and
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middle) and two internal locations a third of the total length of each scat away from the ends.
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This method yielded approximately 0.1 g of scat that was incubated overnight with lysis
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buffer and Proteinase K. The mixture was then centrifuged at 13,400 RPM for 30 sec and the
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liquid portion aspirated for DNA extraction. The DNA was eluted with 60 μl of molecular
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grade water.
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SI1.2. Sequence identification and primer design
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New primers were designed for this study as published specific leopard cat cytochrome b
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primers by Mukherji et al. (2010) targeting a similar length of the desired region were found
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to be too unspecific to yield PCR products from the scat samples. LC-CB F (5’-CAC GAT
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TCT TCG CTT TCC-3’) and LC-CB R (5’-GAT ATG RGG AGG GGT GTT-3’) were
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designed for this study to amplify a 242 bp region of the Southeast Asian leopard cat
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cytochrome b gene, while new primer pairs LC-CR F (5’-TGC TTG CCC AGT ATG TCC-
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3’) and LC-CR R (5’-GCT GGT TTC TCG AGG CTA-3’) were designed to amplify a 527
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bp region of the control region. Testing of these newly designed primers on Oriental house rat
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and domestic cat DNA were performed to ensure that the primers were specific to targeted
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sites of leopard cat mtDNA.
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SI13. Amplification and sequencing of DNA
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Polymerase chain reaction (PCR) was carried out in 25 μl volumes containing; 2.5 μl 10X
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buffer, 2.0 μl dNTP’s, 1.0 μl of each primer, 2.5 μl BSA (0.1mg/ml), 0.15 μl TaKaRa Ex Taq
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HS (Takara Bio Inc), 1.5 μl of template DNA, and 14.35 μl of molecular grade water. PCR
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was performed in an Eppendorf Master Cycler ep Gradient S (Eppendorf AG) thermal cycler
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using the following touchdown protocol: LC-CB: 94°C for 30 s, 57°C for 45 s, and 72°C for
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50 s for five cycles, followed by 94°C for 30 s, 55°C for 45 s, and 72°C for 50 s for 35
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cycles. LC-CR: 94°C for 30 s, 60°C for 45 s, and 72°C for 50 s for five cycles, followed by
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94°C for 30 s, 58°C for 45 s, and 72°C for 50 s for 35 cycles. A negative PCR control was
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included with each set of PCR reaction. The presence of PCR products was confirmed
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through electrophoresis on 1% agarose gels in TAE buffer in presence of EtBr. Gels were
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visualized under ultraviolet light, and a successful PCR reaction was defined as one
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producing specific product in the expected size range.
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The PCR products were purified using SureClean (Bioline) according to manufacturer’s
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instructions with the following modifications: 1) 100 μl of 70% ethanol was added to the
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sample and vortex for 30 seconds for the washing step, 2) the final elution was carried out
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with 20 μl water. Cycle sequencing was performed using BigDye Terminator PCR (Applied
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Biosystems) in both directions following manufacturer’s instructions. The resulting single-
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stranded DNA were purified with CleanSEQ magnetic beads (Agencourt Bioscience Corp),
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and sequencing was performed on an ABI 3100xl genetic analysis sequencer (Applied
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Biosystems).
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SI1.4. Sequence alignment and haplotype determination
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Sequences were edited and assembled with SEQUENCHER 4.5 (Gene Codes Corporation).
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The same software was used to align edited mtDNA sequences. Sequences with ambiguous
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base calls, usually due to overlapping nucleotide peaks or indistinct signal were re-sequenced,
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or if necessary, re-extracted, PCR amplified and sequenced again. Samples that did not
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contain leopard cat hair, and repeatedly yielded ambiguous sequences that were not
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diagnostic of leopard cat sequences after re-extraction were not included in the diet analyses.
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Sequences were verified to be of leopard cat origin by 1) presence of a clear band in on the
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gel after PCR, and 2) match with leopard cat entries for the relevant mtDNA markers in
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GenBank using BLAST (National Center for Biotechnology Information, Bethesda, MD).
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