Cleanup standard kit

PCR was performed on individual bacterial colonies using a ScreenMix-HS reaction mixture (Evrogen, Moscow, Russia) in an Eppendorf MasterCycler Personal cycler. Direct and reverse primers for bacterial DNA amplification were 785F (5′-GGATTAGATACCCTGGTA) and 1492R (5′-TACGGYTACCTTGTTACGACTT) [17 (link)], respectively. PCR was performed in the following regime: Denaturation at 94 °C for 4 min, 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s, and final elongation at 72 °C for 10 min. Amplified fragments were isolated from the gel and purified with a Cleanup Standard kit (Evrogen, Moscow, Russia). Purified PCR products were sequenced with an Applied Biosystems 3500 sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit, and obtained nucleotide sequences were analyzed using the GenBank database. Organisms were assigned to certain taxa if their DNA sequences coincided by no less than 99% with the corresponding sequences already deposited in the databases by at least five different authors.

PCR amplification of fragments for genomic integration and cloning was performed using Q5^® High-Fidelity DNA Polymerase (New England Biolabs (Ipswich, MA, USA), M0491). DNA fragments were purified from the PCR mix using a Cleanup Standard Kit (Evrogen (Moscow, Russia), BC022). PCR for checking colonies was carried out using Taq DNA Polymerase (ThermoFisher, EP0401). Total DNA of yeast strains was isolated using a standard method [13 (link)]. Plasmids maintained in E. coli were isolated using the Monarch^® Plasmid Miniprep Kit (New England Biolabs, T1010).

The DNA sequences of the humanized variable domains of the hB16 antibody were synthesized by a chemo-enzymatic method (Evrogen, Moscow, Russia). Fragments that encode light-chain variable domain and heavy-chain variable domain sequences of humanized hB16 antibody were generated by PCR, separated from other reaction products by DNA-electrophoresis and purified on CleanUp standard kit (Evrogen, Russia) columns. The nucleotide sequences of VL and VH were joined with Ck and CH1-CH3 nucleotide sequences of human IgG1 by SOE-PCR, digested with NheI and XhoI endonuclease restriction enzymes and cloned into a pcDNA3.4 vector.

Genomic RNA from immunocaptured virus particles was employed for synthesis and amplification of cDNA libraries using Complete Whole Transcriptome Amplification Kit (WTA2, Sigma-Aldrich, St. Louis, MO, USA) as described previously [40 (link)]. The resulting libraries were subjected to the high-throughput sequencing on the Illumina MiSeq platform (isolates Ka7, Ka15, Ka23) or the 454 platform (Bg6, Bg26, Bg66, Pul). The contigs were assembled de novo using the SPAdes v.3.10.1 program [41 (link)]. The PPV-relevant contigs were identified by a BLASTn search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and aligned on the PPV genome that showed the greatest BLAST score. Appropriate primers were designed to fill the internal gaps. The 5’-terminal region was amplified using 5’RACE kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. The corresponding PCR products were purified from agarose gel with Cleanup Standard kit (Evrogen) and sequenced in both directions using Evrogen facilities. The full-length genome sequences were deposited in GenBank under accession numbers MH311853–MH311859.

Genomic DNA was isolated from the monolayer cell culture using a commercial column-based DNA extraction kit (ExtractDNA Blood kit, Evrogen). To confirm the presence of c.1252G>T mutation in patient-derived fibroblasts and iPSCs, or to analyze DNA changes in CRISPR/Cas9-edited iPSC clones, the target region of exon 9 in the MEN1 gene was amplified by PCR from genomic DNA using a pair of primers (forward 5′-TACGGGATTAGGGATGGCAG-3′, reverse 5′-GGGCCAGAAAAGTCTGACAA-3′). The primers used to examine off-target effects in CRISPR/Cas9-edited iPSC clones are shown in Table S5. The PCR products were purified using a Cleanup Standard kit (Evrogen) followed by Sanger sequencing (Evrogen).

High‐molecular‐weight DNA was isolated from fin tissue preserved in ethanol using a QIAamp DNA Mini Kit (Qiagen) or obtained with a salt‐based DNA extraction method (Aljanabi & Martinez, 1997 (link)) followed by purification using a CleanUp Standard kit (Evrogen). The quantity of dsDNA was measured using a dsDNA HS Assay Kit for fluorometer Qubit 3 (Life Technologies). A ddRAD‐library was constructed following the quaddRAD protocol (Franchini et al., 2017 (link)) using restriction enzymes PstI and MspI. In total, 63 DNA samples of Labeobarbus ecomorphs from five riverine basins (see Table S1) were sequenced by two runs of Illumina HiSeq2500 and Illumina X Ten (2 × 150 bp paired‐end reads). The raw sequencing data from 63 samples were demultiplexed by the sequencing provider using outer Illumina TruSeq dual indexes and deposited at NCBI (BioProject ID PRJNA1000117).

DNA from the grown colonies was isolated using commercially available PROBA-GS kit (DNA Technology, Russia). Polymerase chain reaction was performed with an Eppendorf MasterCycler Personal cycler. Each PCR reaction mixture contained 2.5 µl of 10× reaction buffer, 1 µl of 10 mM dNTPs, 1 µl of 10 µM forward primer, 1 µl of 10 µM reverse primer, 3 µl of 25 mM Mg²⁺, 1 µg of template DNA, 2.5 units of thermostable Taq DNA polymerase (Evrogen, Russia), and deionized water (up to 25 μl). PCR regime included initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, elongation at 72 °C for 45 s; final elongation at 72 °C for 10 min. Fungal-specific primers for molecular identification were: direct ITS1 primer—5′-TCCGTAGGTGAACCTGCGG; reverse ITS4 primer—5′-TCCTCCGCTTATTGATATGC (White et al. 1990 ).
PCR products were stained with ethidium bromide and visualized at 312 nm with a TCO-20LM transilluminator after electrophoresis in 2% agarose gel.
RCR products were purified from the gel with a Cleanup Standard kit (Evrogen, Russia) and sequenced with an Applied Biosystems 3500 automated sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit and ITS1/ITS4 primers.