The total cellular RNA was extracted from transfected or infected cells (from 6-well plates each) with RNeasy Mini kit (Qiagen, Wrocław, Poland) according to manufacturer’s instructions. The cDNA was generated with Maxima H minus First Strand cDNA synthesis kit (Thermo, Poland) according to the manufacturer’s instructions with 2 µg of RNA and oligoT primer. The obtained cDNA was subjected to PCR reaction with EAVFor and EAVRev primers (each 10 mM) listed in Table 1 and one-fusion high-speed-fidelity polymerase (GeneON, Abo, Poland). The thermal profile was as follows: initial denaturation at 98 °C for 5 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 46 °C for 20 s and extension at 72 °C for 20 s, and a final extension at 72 °C for 3 min. The RT-PCR products were gel-purified using a QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA), and the sense and antisense strands were sequenced (Eurofins MWG Operon, Huntsville, AL). The sequence data were analyzed using the software FinchTV 1.5.0. PCR products were subjected to agarose gel electrophoresis, purified (Gel-Out, A&A Biotechnology, Gdynia, Poland), and the sense and antisense strands were sequenced (Genomed, Warsaw, Poland).
Gel out
Manufactured by A&A Biotechnology
Sourced in Poland
Gel-Out is a laboratory equipment designed for the extraction and purification of DNA or RNA from agarose gels. It is a versatile tool used in molecular biology and biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Midori green dna stain, by Nippon Genetics (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Midorigreen dye, by Nippon Genetics (1 mentions)
Mj mini gradient pcr apparatus, by Bio-Rad (1 mentions)
Quick blood dna purification kit, by EURx (1 mentions)
Phire hot start 2 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Plasmid mini, by A&A Biotechnology (1 mentions)
Noti enzyme, by Thermo Fisher Scientific (1 mentions)
Total rna midi, by A&A Biotechnology (1 mentions)
Taq dna polymerase, by A&A Biotechnology (1 mentions)
5 protocols using gel out
1
RT-PCR and Sequencing of Viral RNA
2
Nested PCR for Screening of aHEV Samples
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For the screening of aHEV-positive samples, the nested polymerase chain reaction (PCR) assay was performed with two degenerate primers sets that targeted the partial helicase gene region in ORF1, as described previously [13 (link), 17 (link)]. A previously sequenced aHEV fragment was used as a positive control (GenBank accession number MH636899.1). The size of the obtained PCR product was 386 bp.
The products resulting from second-round PCR were examined on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Düeren, Germany). The amplified products of ORF1 were excised, purified using Gel-Out (A&A Biotechnology, Gdynia, Poland), and directly sequenced in both directions with Sanger’s method (Eurofins Genomics Sequencing GmbH, Cologne, Germany) with the use of PCR primers.
The products resulting from second-round PCR were examined on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Düeren, Germany). The amplified products of ORF1 were excised, purified using Gel-Out (A&A Biotechnology, Gdynia, Poland), and directly sequenced in both directions with Sanger’s method (Eurofins Genomics Sequencing GmbH, Cologne, Germany) with the use of PCR primers.
3
Recombinant Expression of TtCutA Variants
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The plasmids and oligonucleotides used in this study are listed in Supplementary Tables S1 and 2 , respectively. All restriction enzymes and Phusion DNA polymerase for the amplification of inserts were from Thermo Fisher Scientific. DNA purification kits (DNA Plasmid Mini and Gel-Out) were from A&A Biotechnology.
The pCC19-pCC32 plasmids used for the expression of truncated TtCutA variants were generated by sequence- and ligation-independent cloning (SLIC) (35 (link)) in E. coli MH1 strain. Inserts were amplified using respective primers listed inSupplementary Table S2 and were cloned into BamHI-XhoI sites of the pET28M N-6xHis-SUMOTag vector.
The pCC33, pCC34, pCC35, pCC36 and pCC37 plasmids for the expression of TtCutA240–603 truncation variants with single amino acid substitutions were generated by site-directed mutagenesis with oligonucleotide pairs: mutN397Af-mutN397Ar, mutA400Gf-mutA400Gr, mutN403Af-mutN403Ar, mutR557Af-mutR557Ar and mutR557Hf-mutR557Hr, respectively, using pCC25 as the template. The presence of introduced mutations was verified by sequencing the inserts with appropriate primers, listed inSupplementary Table S2 .
The pCC19-pCC32 plasmids used for the expression of truncated TtCutA variants were generated by sequence- and ligation-independent cloning (SLIC) (35 (link)) in E. coli MH1 strain. Inserts were amplified using respective primers listed in
The pCC33, pCC34, pCC35, pCC36 and pCC37 plasmids for the expression of TtCutA240–603 truncation variants with single amino acid substitutions were generated by site-directed mutagenesis with oligonucleotide pairs: mutN397Af-mutN397Ar, mutA400Gf-mutA400Gr, mutN403Af-mutN403Ar, mutR557Af-mutR557Ar and mutR557Hf-mutR557Hr, respectively, using pCC25 as the template. The presence of introduced mutations was verified by sequencing the inserts with appropriate primers, listed in
4
Pigeon Genomic DNA Extraction and A4GALT Gene Analysis
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Genomic DNA was isolated from livers of sacrificed pigeons using Quick Blood DNA Purification Kit (EURx, Gdansk, Poland) according to the protocol provided by the supplier. Primers and conditions used in the PCR reaction for amplification of the A4GALT gene are listed in Supplementary Table S3A,B . PCR was performed using an MJ Mini gradient PCR apparatus (Bio-Rad, Hercules, CA, USA) in 100 µL reaction mixes containing 200 ng of pigeon genomic DNA (template), 0.2 mM dNTPs, HF Taq buffer with MgCl2 (20× dilution), 0.2 mM forward and reverse primers, and 1 unit of HF Taq polymerase (Thermo Fisher Scientific, USA). The PCR products were purified using Gel-Out (A&A Biotechnology, Gdansk, Poland). The PCR products were further amplified using primers with restriction sites: XhoI (F) and NotI (R) (Thermo Fisher Scientific, Waltham, MA, USA) and cloned into the pCAG vector (kindly provided by Prof. Peter W. Andrews, University of Sheffield, Sheffield, UK) [18 (link)]. To test for the presence of A4GALT paralogs, the PCR products were digested with PaeI and HincII (Thermo Fisher Scientific, Waltham, MA, USA); 500 ng of purified PCR products were digested in 37°C for 6 h and analyzed by electrophoresis in 1% agarose gel with MIDORI green dye (Nippon Genetics Europe, Duren, Germany).
5
Standard Molecular Biology Protocols
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Standard molecular biology protocols used in this study followed the methodologies described in [60] . All oligonucleotides and longer synthetic DNA fragments used in this study are listed in Additional File_2. E. coli and Y. lipolytica transformations were conducted according to standard heat-shock protocols described in [60] and [61] . Total RNA isolation from Y. lipolytica cells, plasmid isolation from E. coli, DNA fragments extraction from agarose gel or purification of DNA fragments were all conducted using appropriate kits from A&A Biotechnology (Gdynia, Poland) -Total RNA Midi, Plasmid Mini, Gel-Out or Clean-Up. Restriction digestion of DNA fragments was done using either NotI enzyme (Thermo Fisher Scientific, Waltham, USA) or BsaI (New England Biolabs, Ipswich, USA). Routine colony PCR with E. coli biomass was run using Taq DNA polymerase (A&A Biotechnology), while colony PCR with Y. lipolytica biomass was conducted using Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific). All the reactions and protocols were conducted according to the manufacturers' recommendations.
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