Phusion hot start 2 dna polymerase

For the identification of birds, mammals, reptiles and fish, a 303 bp fragment of mtDNA cox1 gene was PCR-amplified with primers AVS2F and AVS3R as described in Tull et al. (2022 (link)). PCR reactions were carried out in a total volume of 20 μL with 1× Phusion HF Buffer (Thermo Fisher Scientific), 0.2 mm dNTP, 0.25 μm of each primer and 0.4 U Phusion Hot Start II DNA Polymerase and 2 μL of purified DNA. The PCR mixture was initially denatured at 98°C for 30 s, followed by 10 touchdown cycles for 10 s at 98°C, 20 s at 60°C (reducing the temperature 1°C per cycle) and 30 s at 72°C, followed by 30 cycles of 10 s at 98°C, 20 s at 50°C and 30 s at 72°C. In case the PCR was negative due to highly degraded DNA, we performed a second analysis by PCR-amplifying a shorter, 183 bp fragment of mtDNA 12S rRNA gene, using primers Ave12F and Ave12R, described in Oja et al. (2017 (link)). PCR products were checked using 2% 1×TAE gel-electrophoresis and visualized under UV radiation using ethidium bromide.
PCR products were purified, sequenced and nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify various taxa, such as reptiles, fish and birds.

For the MEDBlack cruise DNA samples, the hypervariable V4-V5 region of the 16S rRNA gene from Bacteria and Archaea was amplified with high-fidelity Phusion Hot Start II DNA polymerase (Thermo Scientific, Waltham, MA, USA) using universal primers 515F and 928R (34 (link)) and a two-step PCR protocol (35 (link)), as detailed in Supplemental Information 1.2. and Table S1. The correct amplicon size and the absence of non-specific bands were checked by agarose gel electrophoresis. Amplicons were sequenced using a 2 × 250 bp paired-end MiSeq system (Illumina, USA) at the Genotoul platform (Toulouse, France). The raw sequences have been deposited at NCBI GenBank, SRA database, under the BioProject accession number PRJNA895066.
Raw sequences were analyzed on the Galaxy bioinformatics platform through the FROGS pipeline, version 3.2.3, as detailed in the Supplemental Information. Especially, operational taxonomic units (OTUs) were defined by sequence clustering, using the high-resolution SWARM algorithm v3.2.3 (36 (link)). After filtering at 0.005% of abundance, OTUs were taxonomically annotated with the SILVA 16S database (version 138.1).
Bacterial and archaeal abundances were quantified in the MEDBlack cruise DNA samples by quantitative PCR with Takyon No Rox SYBR 2X Master Mix (Eurogentec, Seraing, Belgium). Protocol details are provided in Table S1.

Either Taq DNA Polymerase (Thermo Fisher), KOD DNA Polymerase (Merck Millipore), or Phusion Hot Start II DNA Polymerase (Thermo Fisher) was used for gene amplifications according to the manufacturer’s instructions. Primers were from IDT and restriction enzymes from Thermo Scientific (Fast Digest). NucleoSpin Gel and PCR Clean-up (Macherey and Nagel) was used for isolating DNA fragments from agarose gels. T4 DNA ligase was from New England Biolabs. PCR amplicons were routinely cloned to pGEM-T Easy (Promega) and sub-cloned to lactococcal plasmids. Plasmid DNA was isolated with NucleoSpin Plasmid (Macherey and Nagel), with an additional lysozyme treatment step for L. lactis. Lactococci were transformed with electroporation using a Gene Pulser II apparatus (Biorad) according to the MoBiTec GmbH (Goettingen, Germany) instructions. All plasmids were sequenced by GATC (Constance). All primers and plasmids are listed in Supplementary Table S1.

VviAGL11 gene sequences (promoter and CDS) were amplified from genomic DNA of table grapevine varieties by using the Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific). PCR products were purified from agarose gel using Illustra GFX PCR DNA and Gel Band Purification Kit (Merck KGaA, Darmstadt, Germany) and then sequenced by Ion Torrent technology (Thermo Fisher Scientific). Amplicon libraries were prepared by using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific) following manufacturer instructions. The enriched library was loaded on Ion PGM 314 chip and sequenced on the Ion PGM™ sequencer using Ion PGM™ Sequencing 200 Kit v2. Reads were aligned on the grapevine reference genome (12X.v2) of Pinot Noir [43 (link)]. The depth of sequencing obtained was approximately 800X. Variant caller plugin provided in the Torrent Suite Software was run for the identification of polymorphisms across the reference. Sequences were viewed by the Integrative Genomics Viewer (IGV) [44 (link)].

MCF-7 cells, obtained from the cell repository of the University of Freiburg, Germany, were cultured in DMEM containing 10% FCS at 5% CO₂ at 37°C and 95% humidity. Cells were seeded on 24 well plates and cultured for 2 days (yielding subconfluent cell layers with approximately 50% confluence) or 5 days (yielding confluent cell layers). After stimulation with Dex for different time periods, the cells were washed with PBS, trypsinized, and resuspended in DMEM. The pellet was washed two times with PBS before mRNA extraction with the Quick-RNA MiniPrep Kit (Zymo Research); cDNA synthesis was performed with the ProtoScript II Reverse Transcriptase (NEB), both according to manufacturer's instructions. AQP5 and GAPDH expression were quantified by qPCR with the qAQP5_human and qGAPDH_human primers, respectively, using 0.25 U Phusion HotStart II DNA Polymerase (Thermo Scientific) with an initial heating step of 98°C for 30 sec followed by 45 cycles at 99°C for 5 sec, 63°C for 15 sec, and 72°C for 5 sec (see primer sequences in Supplementary Table  1). Primers were designed to anneal in conserved regions between humans and mice; therefore, the same plasmids could be used as standards (10⁷, 10⁶, 10⁵, 10⁴, and 10³ molecules/reaction) for measuring murine or human absolute AQP5 mRNA levels.

Genomes of the 20 S. halichoeri isolates were sequenced at the Institute of Biotechnology (University of Helsinki, Finland) using next-generation sequencing platforms. Genomic DNA (0.5 mg) was sheared using a Bioruptor NGS Sonicator (Diagenode) to approximately 600 bp fragments. The fragments were polished, A-tailed, and ligated to a TruSeq truncated adapter. Purification of the ligation reaction was done using AMPure XP beads (Agencourt, Beckman Coulter). PCR of the libraries were done using Phusion Hot Start II DNA Polymerase (Thermo Fisher) and index P7 primers and full-length P5 adapter primers. The reactions were pooled and purified with AMPure XP beads. Size selection of the pool was done according to Lundin et al. [11 (link)]. The obtained library pool was paired-end sequenced on a MISeq Sequencer using the v3 600 cycle kit (Illumina).
Genomes of the 20 newly sequenced S. halichoeri strains were deposited in GenBank under the accession numbers listed in Table 1. The annotation was performed using the assembled DNA sequences of the 20 new draft genomes from these isolates. The genomes were run through an automatic annotation pipeline RAST (Rapid Annotation using Subsystem Technology) [12 (link)], followed by manual curation in few cases.