Cfr13I (Sau96I) (10 U/µL)

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	The URA5-RFLP technique was conducted according to Meyer et al. (2003) PCR products were double digested with Cfr13I (Sau96I) and HhaI enzymes (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h and separated in 3% agarose gel at 100 V for 3 h.

Protocol tips
The URA5-RFLP technique was conducted according to Meyer et al. (2003) PCR products were double digested with Cfr13I (Sau96I) and HhaI enzymes (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h and separated in 3% agarose gel at 100 V for 3 h.

Publication protocol

Environmental samples were collected in the form of swabs taken from tree holes and pigeon droppings, or obtained directly from pigeons. Specimens obtained directly from birds (throat, crop and cloaca swabs) were collected during the annual, routine clinical examinations of health status in pigeon flocks. Both the tree and excreta samples were placed in tubes containing 3 mL of sterile saline solution, vortexed for 2 min and left for 10 min in order to let the suspension settle. Then the supernatants were diluted 1:10 with saline. Two sets of plates containing Niger seed agar (NSA) were inoculated with 100 μL of the supernatant and its dilution, respectively. The swabs taken from birds were inoculated directly onto the NSA. Plates were incubated at 30 °C for up to 14 days, then colonies showing different degrees of melanisation were subcultured in order to purify the cultures. The isolates were tested using India Ink staining and prepared for further studies. DNA extraction was performed using the MasterPure™ Yeast DNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA), according to the manufacturer’s instructions. The DNA of tested strains was analysed using multilocus sequence typing (MLST) and/or URA5-RFLP methods. MLST analysis was performed according to the ISHAM consensus scheme concerning seven genetic loci (CAP59, GPD1, LAC1, PLB1, SOD1, URA5, and IGS1) (Meyer et al. 2009). For amplification of the SOD1 gene of C. neoformans var. neoformans strains, an alternative reverse primer was used, as described in Sanchini et al. (2014); PCR conditions for LAC1 locus, presented by Litvintseva et al. (2006), were applied instead of those given in the consensus. PCR products were purified and sequenced (DYEnamic ET terminator cycle sequencing kit ABI Prism™, Amersham Biosciences Europe GmbH, Germany). Forward and reverse sequences were assembled using BioEdit v7.2.0 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and then analysed by BioloMICS Polyphasic Identification Software (http://mlst.mycologylab.org/cneoformans); as a result, allele types (AT) of each gene as well as sequence types (ST) and molecular types (MT) of the tested strains were assigned. The URA5-RFLP technique was conducted according to Meyer et al. (2003) PCR products were double digested with Cfr13I (Sau96I) and HhaI enzymes (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h and separated in 3% agarose gel at 100 V for 3 h. RFLP patterns of the tested strains were determined visually by comparison with standard strains (CBS 8710-VNI, CBS 10084-VNII, CBS 132-VNIII and CBS 10079-VNIV). Restriction maps of the tested and reference strains were prepared using BioEdit v7.2.0 software. As a reference, a sequence of the URA5 gene of the WM 629 (CBS10079) strain was used. Since a sequence matching the exact length of the PCR product obtained in the URA5-RFLP method was not available in GenBank, a shorter sequence was chosen (GenBank accession number KC568724). Sero- and mating-types of all tested isolates were established using a PCR-based method of amplification of serotype-specific and mating-type-specific STE20 gene, described by Li et al. (2012). The following strains were used as positive controls: CBS 10084 (Aα), CBS 132 (AαDa), IUM 96–2828 (Aa) and CBS 10079 (Dα). Additionally, primers specific for LAC1 locus of A or D types, designed by the same authors, were used to enable discrimination of a serotype (Li et al. 2012).

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