Powerclean pro dna clean up kit

P. cutis JCM 15793 and P. stagnora JCM 9641 were obtained from the Japan Collection of Microorganisms, RIKEN BioResource Center (RIKEN BRC-JCM), Japan. P. cutis was cultured in 250 mL of YM broth (1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, Difco) for 3 days at 30 °C under constant shaking (150 rpm), and the cells were collected by centrifugation. P. stagnora was cultured on YM agar at 25 °C for 10 days, followed by collecting the cells by scraping. The cell mass was freeze-dried, and ground in a mortar. Total DNA was extracted using phenol/chloroform/isoamyl alcohol, precipitated by adding 2-propanol, and then spooled out with a sterile glass rod. The crude DNA was dissolved in G2 Buffer (Qiagen, Cat. No. 1014636), and purified using a Genomic-tip 100/G (Qiagen, Cat. No. 10243) according to the manufacturer’s instruction. The DNA was further cleaned using PowerClean Pro DNA Clean-Up Kit (MO Bio Laboratories, Cat. No. 12997-50) and used for the library preparation for subsequent sequencing.

Silica‐desiccated samples had DNA extracted with a NucleoSpin^® Plant II DNA kit (Macherey‐Nagel) following the manufacturer's protocol with a potassium acetate (KOAc) addition for extracting from polysaccharide‐rich algae (e.g., Saunders 1993 , Dos Reis Falcão et al. 2008 ). Unpurified DNA was cleaned with a PowerClean^® Pro DNA Clean‐Up Kit (Mo Bio Laboratories, Carlsbad, CA, USA) and DNA purity was assessed with a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Frozen Lympha mucosa samples for total RNA isolation were processed within 4 months of collection to reduce the potential for sample degradation. Total RNA was isolated using a NucleoSpin^® RNA Plant Kit (Macherey‐Nagel) following the manufacturer's protocol with KOAc addition as above, and DNA digestion. However, for RNA samples, the KOAc incubation period was performed at −20°C, followed by 4°C centrifugation at 12,000g for 10 min to reduce RNA degradation. RNA concentration and purity were assessed using a NanoDrop Spectrophotometer, and an Agilent 2100 Bioanalyzer. RNA samples were stored in a freezer of −80°C.

Fecal pellets were collected upon handling into an autoclaved microtube, placed on wet ice and stored at -80°C. Bacterial DNA was extracted with QIAamp DNA Stool Mini Kit 51504 (Qiagen). Quality and quantity of DNA were spectrophotometrically evaluated (NanoDrop 1000, Thermo Scientific, USA) and stored at -80°C. Samples were cleaned (PowerClean^® Pro DNA Clean-Up Kit, Mobio Laboratories, USA), and spermine was added to prevent DSS inhibiting the polymerase (unpublished manuscript).

A total of 14 soil samples were collected (7 per carcass). DNA was isolated in two replicates from each sample (P1 and P2) resulting in a total of 14 DNA samples per carcass site. DNA was isolated using the FastDNA® spin kit for soil (MP Biomedicals, Santa Ana, California, USA), following the manufacturer’s protocol with adjustments specified in Additional file 4. The DNA samples were filter sterilised using an Ultrafree® Durapore PVDF 0.1 μM spinfilter (Millipore, Darmstadt, Germany).
DNA was shipped dry, following an ethanol precipitation, from ENP to Norway (see Additional file 4: for details). Upon arrival DNA was re-suspended in DES buffer from FastDNA® spin kit for soil. Samples were concentrated and purified using Agencourt® AMPure® XP beads (Beckman Coulter, Beverly, Massachusetts, USA), and treated with a PowerClean® Pro DNA clean-Up Kit, (MO BIO Laboratories, Carlsbad, California, USA) to remove any remaining inhibitors.

DNA was isolated from 0.5 g sediment incubations aseptically in a sterile laminar flow hood using a sterile spatula, and extractions were carried out as described previously (Pichler et al., 2018). Purification of DNA extracts was carried out with the PowerClean Pro DNA Clean‐up Kit (MO BIO Laboratories) and DNA was quantified with the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific) according to manufacturer's instructions. For preparation of fungal mock community ITS1 sequencing libraries, 13 different freeze‐dried cultures (Table 1) were purchased from the Leibniz‐Institute DSMZ‐German Collection of Microorganisms and Cell Cultures, rehydrated in lysogeny broth (LB) following the manufacturer's instructions and grown on agar plates (yeast extract‐peptone‐dextrose [YPD] broth) for 3–7 days at room temperature. DNA was extracted using a previously described protocol (Pichler et al., 2018). Three technical replicates of the mock community were created to assess variation in Illumina sequencing fidelity on the MiniSeq platform across two different sequencing runs. DNA from laboratory dust and extraction blanks was extracted and purified in order to identify and remove any contaminant sequences introduced during the laboratory processing of the samples.

Three sediment samples, denoted N1, N4, and N5, were collected from Qinghai Lake, a perennial salt lake located in a structural intermontane depression at the northeastern corner of the Qinghai–Tibetan Plateau (54 (link), 55 (link)), China, in June 2018. Each sample was placed in a sterile plastic bag using a sterile scoop and immediately stored at 4°C. The GPS coordinates of the sampling sites are listed in Data Set S1, Sheet 1. A fraction (10 g by wet weight) of each sample was subjected to DNA extraction as described previously (23 (link), 56 (link)). DNA was purified by using a PowerClean Pro DNA Clean-Up Kit (MO BIO Laboratories), sheared to 400~500 bp in size with the Covaris M220 Focused-Ultrasonicator and quantified by the Agilent 2100 bioanalyzer (Agilent Technologies Inc., USA). The sequencing libraries were constructed using the KAPA Hyper Prep Kit (Kapa Biosystems). Paired-end (PE) sequencing (2 × 250 bp) was conducted on an Illumina Hiseq-2500 platform at Beijing Institute of Genomics, Chinese Academy of Sciences (CAS), Beijing, China. Deep-sea sediments (TVG05 and TVG06), from the Southwest Indian Ocean during the DY125-39 cruise with R/V Dayang No.1, have been previously described by Zheng et al. (23 (link)).