Nzygelpure kit

The internal transcribed spacer (ITS) region of nuclear rDNA was amplified through PCR from genomic DNA by using ITS1 and ITS4 primers [44 ]. PCR reactions consisted of 1 μL of gDNA, 1.5 mM MgCl₂, 0.2 mM dNTPs (Fermentas, Thermo Scientific, Waltham, MA, USA), 1 mM of each primer, and 2.5 U of Dream-Taq DNA polymerase (Fermentas, Thermo Scientific, Waltham, MA, USA) in a total reaction volume of 50 mL. Amplification was carried out in a Thermal Cycler (Bio-Rad, Hercules, CA, USA) at 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 55 s, and 72 °C for 2 min and a final extension at 72 °C for 10 min. Amplified products were analyzed by 1% agarose gel electrophoresis prepared with 0.5 × Tris-Borate-EDTA buffer (TBE) and visualized on a Gene Flash Bio Imaging system (Syngene, Cambridge, UK). PCR reaction products were purified using the NZYGelpure kit (Nzytech, Lisbon, Portugal), according to the manufacturer’s protocol. Samples were quantified using a spectrophotometer NanoDrop-2000C (Thermo Scientific, Waltham, MA, USA) and sequenced in a sense strand by Macrogen Inc. (Madrid, Spain). ITS sequence homology was explored at the National Center for Biotechnology Information (NCBI) database using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/) (BLASTn). All the fungal sequences that showed resemblance to at least 91% were used to identify the fungus analyzed.

For the selective amplification of a fragment sequence of the 16S recombinant deoxyribonucleic acid (rDNA) of Ca. Erwinia dacicola, a specific primer (EdF1) was paired with 1507R for PCR as previously described [11 (link)]. PCR reactions were conducted using 1 μl of the extracted DNA in a standard 25 μl reaction, with 0.5 pmol/μl of each primer, 1.5 mM MgCl², 0.5 mM dNTPs and 0.04 U/ml Taq DNA polymerase with the following cycle protocol: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing for 30 s at 55 °C and extension at 72 °C for 30 s, followed by a final extension of 72 °C for 10 min. The PCR products, after visualized on agarose gel, were purified using the NZYGelpure kit (from NZYTech, Lda) and sequencing was done commercially (Macrogen Inc.). Electropherograms were inspected using Genestudio (www.genestudio.com). The evolutionary history was inferred by Maximum Likelihood and using the Hasegawa-Kishino-Yano model as selected model of evolution. Both analyses were performed in Mega X [25 (link)].

To analyse relative allelic expression of the H19, Meg3 and Snrpn imprinted genes, cDNA was generated as described in RT-qPCR and amplified by PCR using the primers in Supplementary Table 2. The PCR product was cleaned using the NZYGelpure kit (Cat# MB01102, Nzytech) and sent for Sanger sequencing to STABVIDA sequencing company and data were visualised and analysed on a Chromas v2.6.2 software.

Insertion of a kanamycin resistance cassette in the pUA1139 plasmid was performed using a λ-Red recombinase strategy (Datsenko and Wanner, 2000 (link)). Briefly, the kanamycin resistance cassette from pKD4 (Supplementary Table S1) was amplified by PCR using the primers with homology with the desired regions (Supplementary Table S2). The resulting linear PCR products were purified with NzyGelpure kit (NZYTech, Lisboa, Portugal), and were introduced by electroporation in the target bacteria carrying the plasmid pKOBEGA (Ap^R; Supplementary Table S1). Transformant clones were selected in LB agar plates supplemented with kanamycin (50µg/mL). The kanamycin cassette insertion was confirmed by PCR and sequencing using the appropriate primers (Supplementary Table S2).

Rapid DNA extraction with chloroform was realised for each transformant and PCR was performed to verify the plasmid transformations and nblA deletion. NZYTaq II 2 × Green Master Mix (Nzytech) was used for all PCR confirmation reactions. The set composed of Oligo 7/Oligo 8 was used to check for Cm^R gene in pSEVA351 and derived plasmids, the AL040/AL041 set was used to check for the Spt^R gene in pSEVA451 and derived plasmids, the AL034/AL035 set was used to check for the Km^R gene in pSL2680 and derived plasmids, and the AL036/AL037 set was used to check for the deletion of nblA on Synechocystis 6803 chromosome. In nblA edited strains, PCR fragments were purified using NZYGelpure kit (Nzytech) and sequenced to confirm the accurate edition (Eurofins Genomics).

After BPW enrichment, 100 µL and 10 µL were spread on Tryptone Bile X-glucuronide agar plates (TBX) and Simmons citrate agar + inositol (SCAi) with and without colistin (3.5 mg/L) and incubated (TBX at 37 °C for 24 h; SCAi at 37 °C for 48 h) for E. coli and Klebsiella spp. detection, respectively. From each plate, between one and five colonies of each morphotype were spread on a CLED medium for further identification by matrix-assisted laser desorption-ionisation-time of flight mass spectrometry (MALDI-TOF VITEK MS, bioMérieux) and standard PCRs for E. coli and K. pneumoniae [20 (link)]. Colistin resistance genes (mcr-1 to mcr-5 and mcr-6 to mcr-9) were identified in E. coli, K. pneumoniae and S. enterica isolates using a multiplex PCR published previously [21 (link)]. Amplified simplex PCR products were purified using the NZYGelpure kit (NZYTech, Lisbon, Portugal) and sequenced at Eurofins Genomics (Konstanz, Germany).

When needed, amplicons were purified using the NZYGelpure kit (Nzytech, Lisboa, Portugal). Sequencing was carried out by the ABI Prism system (Applied Biosystems, Waltham, MA, USA) with the Big Dye Terminator V3.1 cycle sequencing kit and the 48-capillary ABI 3730 DNA analyzer. The quality of the sequences was evaluated with BioEdit software v7.0.0 [33 ] and the characterization of the genogroup and the genotype of the sequenced viral capsid genes was carried out based on NCBI-BLAST data. The sapovirus sequences obtained in this study were deposited in GenBank under accession numbers MT580810, MT58081, MT580812–MT580814, MT580815–MT580861, MT580862–MT580882, MT580883–MT580890, MT580891–MT580896, for the partial capsid sequences.
Sequences were aligned and analyzed with software programs Clustal X 2.0 [34 (link)], GeneDoc 2.7 and MEGA7 (Molecular Evolutionary Genetics Analysis v7.0) [35 (link)]. Phylogenetic trees were built by the maximum-likelihood method (bootstrap of 1000 replicates), with the sequences from this study and reference sequences retrieved from GenBank. Genotypes were assigned based on BLAST analyses and clustering with reference strains.