Nucleospin tissue xs kit

This study was approved by the internal review board (KWNUIRB-2019-12-004-001), and all participants in this study submitted written informed consent and declared to be infected with no skin disease and not to be exposed to antibiotics or antifungals for at least 1 month before sampling. A total of 73 Korean women that are urban population in Seoul, the South Korean capital, were sorted into one of three age groups: (1) 10–29 years (n = 24), (2) 30–49 years (n = 21), and (3) 50–79 years (n = 28). From the 73 women, 146 skin samples were collected (two skin sites per person) by swabbing the forehead (a 4 × 2 cm area of the center) and the palm of the hand using a sterile swab kit (KustoGen Inc., Chuncheon, Korea) after removing their makeup. Metagenomic DNA was extracted from the skin samples using the Nucleospin^® Tissue XS kit (MACHEREY-NAGEL, Düren, Germany). The V3–V4 region of 16S rRNA genes was sequenced on the Illumina MiSeq platform (Macrogen, Daejeon, Korea). The raw pair-ended amplicon sequence reads were deposited in the NCBI Sequence Read Archive under BioProject PRJNA650212.

Our dataset consisted of total 72 samples: 24 fecal samples for each three study species as equally distributed among females and males. The fecal material was not pretreated in any specific way prior to extraction, and the amount was so minimal (approximately 1 × 1 mm) that it was not practical to weigh the samples. The total set of samples was divided into three subgroups each consisting of 24 samples with equal representation of females and males of each study species (each subgroup contained four males and four females per species). One group was processed using ZR Fecal DNA MicroPrep (hereafter abbreviated as ZR; product nr D6012, Zymo Research, Irvine, California, U.S.A.), the second group using NucleoSpin^® Tissue XS Kit (abbreviation NS; product nr 740901, Macherey‐Nagel, Düren, Germany), and the third group with a traditional salt extraction method (abbreviation SE) (see Appendix S1 for detailed salt extraction protocol applied: Aljanabi & Martinez, 1997; Pilipenko, Salmela, & Vesterinen, 2012). We did not measure DNA concentrations, but expected them to be rather low due to the small amount of sample. Moreover, as we amplified both predator and prey DNA, the total DNA concentration as such would not be informative in any case. Thus, we used 1 μl of template DNA regardless of potential differences in the DNA concentrations.

Genomic DNA was extracted from blood and muscle using the GenElute Blood Genomic DNA kit (Sigma-Aldrich, Darmstadt, Germany) and the NucleoSpin Tissue XS Kit (Macherey-Nagel), respectively. Procedures were carried out according to the manufacturer's protocols. Sample quality and DNA concentration were determined via spectrophotometry using a ND-8000 (NanoDrop Technologies, Thermo Fisher Scientific Inc., Wilmington, DE). To ensure the sequence accuracy, DNA was sequenced from PCR replicates. DNA samples were used to assay 16 microsatellite loci length and the mitochondrial D-loop region polymorphisms in order to genetically characterize each group and to clarify the genetic relationships among them.

All tumors were reviewed by a committee of board-certified veterinary pathologists to confirm the histomorphological findings. The selected tissues were ensured to contain more than 90% viable tumor tissue and lacked large areas of hemorrhage, necrosis or autolysis. The tissues were then grossly dissected from three to five 10 μm unstained histological sections. Total genomic DNA was isolated from each specimen using the Nucleospin Tissue XS kit^® (Macherey-Nagel, Bethlehem, PA, USA). Concentration and quality of the DNA was assessed by Qubit 3.0 fluorimeter using the dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA, USA) and TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA), respectively.

DNA recovery from ileal tissue sections was performed as previously described [9 (link)]. Snap-frozen specimens were mounted on appropriate embedding molds (Large, Thermo Scientific #2219; or Small, Sakura Tissue—Tek #4566) with clear OCT compound (Optimal Cutting Temperature Embedding Medium) (Fisher HealthCare #4585) and sectioned with a cryostat instrument (Leica CM 1850 UV, 7 microns). These sections were mounted on membrane slides (Leica PEN—Membrane Slide, 2.0 microns #11505158), stained with toluidine blue (Toluidine Blue 0.1% Aqueous, Newcomer Supply #14027), and air-dried (Sampla Dry Keeper, Samplatec. Corp). DNA was extracted from tissue sections using the Nucleospin Tissue XS Kit (Macherey–Nagel). Quantification of extracted DNA was performed with the KAPA hgDNA Quantification and QC Kit (Roche).