After DNA extraction (Supplementary Material , DNA extraction), a near full-length amplicon (27F/1492R) of the 16S rRNA gene of individual isolates was purified, and sent for Sanger sequencing (LGC Genomics Gmbh, Berlin; Supplementary Material , Sanger sequencing). The sequences were classified with the Ribosomal Database Project Naïve Bayesian Classifier (Wang et al., 2007 (link)), with the 16S rRNA gene training set 16 at a 80% confidence threshold (rdp.cme.msu.edu ), SILVA (Quast et al., 2013 (link)) nr release 138 ACT (Pruesse et al., 2012 (link)) with default options for classification of SSU rRNA genes (https://www.arb-silva.de/aligner/ ) and NCBI Blast. The results were compared, and the most appropriate classification was assigned to the samples.
Sanger sequencing
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Sanger sequencing is a DNA sequencing method used to determine the precise order of nucleotides in a DNA molecule. It is a widely used technique for DNA analysis and is a core function of the lab equipment.
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Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Pwo polymerase, by Roche (2 mentions)
Ficoll hypaque, by GE Healthcare (2 mentions)
Pcdna3, by Thermo Fisher Scientific (2 mentions)
Transcriptor high fidelity cdna synthesis kit, by Roche (1 mentions)
Trizol, by Merck Group (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Clonejet pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Neb hi fi dna assembly kit, by New England Biolabs (1 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Nebasechanger, by New England Biolabs (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
Cd3 apc, by BioLegend (1 mentions)
Cd19 apc, by Thermo Fisher Scientific (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Pcdna3, by New England Biolabs (1 mentions)
30 protocols using sanger sequencing
1
Bacterial 16S rRNA Gene Sequencing and Classification
2
Cloning and Mutagenesis of PPP2CA Cα
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The coding region of wild-type (WT) Cα complementary DNA (cDNA) was cloned into an N-terminal HA-tag eukaryotic expression vector (pMB001) using XbaI/BamHI sites. The mutated PPP2CA construct was directly generated from this plasmid by polymerase chain reaction (PCR)-based site-directed mutagenesis (Stratagene) with Pwo polymerase (Roche Applied Science) and oligonucleotides (IDT) containing the desired point mutations. Forward and reverse primer sequences were 5′-CCATGAGGGTCCAATGCGTGACTTGCTGTGGTC-3′ and 5′-GACCACAGCAAGTCACGCATTGGACCCTCATGG-3′, respectively. Introduction of the variant was verified by Sanger sequencing (LGC Genomics).
3
Identifying and Cloning FhTauT and HsTauT
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Total RNA was isolated from freshly isolated adult flukes using Trizol (Sigma Aldrich, Mannheim, Germany) Reverse transcription was performed using “Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany), using either gene specific primers or oligo dT primers. Starting from a previously deposited cDNA sequence 5’ and 3’ ends of the cDNA were identified via RACE (rapid amplification of cDNA ends) technology, using 5’/3’ RACE Kit, 2nd Generation (Roche Diagnostics GmbH, Mannheim). Recognition sites for XhoI and KpnI were added to the full-length cDNA of FhTauT by PCR. The resulting PCR product was cloned via XhoI and KpnI to peYFP-C1 (Takara Bio Europe, France) generating a transporter tagged with a yellow fluorescent protein at its N-terminus. To generate a non-tagged transporter, FhTauT was also cloned to pcDNA3.1 (Invitrogen, Carlsbad, USA). HsTauT was cloned in a similar way to peCFP-C1 via BamHI and HindIII thereby generating a transporter tagged with the cyan fluorescent protein. Generated sequences were validated by Sanger sequencing (LGC Genomics, Berlin, Germany). The cDNA sequence of FhTauT was deposited at GenBank (NCBI, Bethesda, USA) under the accession number: MG674191.
4
Constructing EHV-1 and EHV-4 gB Plasmids
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Transfer plasmids encoding either EHV-1 or EHV-4 gB with a kanamycin resistance (KanR) gene were constructed. EHV-1 and EHV-4 gB genes were amplified by PCR using primers P1 and P2 or P3 and P4 (Table 1 ). The PCR products were digested with the restriction enzymes XhoI and XbaI (New England Biolabs, NEB, Schwalbach, Germany) and inserted into the vector pBluescript II KS+ (pKS), resulting in recombinant plasmids pKSgB1 and pKSgB4. To construct pKSgB1-KanR and pKSgB4-KanR, the KanR gene was amplified by PCR from plasmid pEPkan-S using primers P5, P6, P7, and P8 (Table 1 ), digested with the appropriate restriction enzymes, and inserted into pKSgB1 and pKSgB4. Correct amplification and insertion were confirmed by Sanger sequencing (LGC Genomics, Berlin, Germany).
5
Agrobacterium-Mediated Transformation of Arabidopsis
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All constructs were verified using restriction digestion and Sanger sequencing (LGC Genomics, Berlin, Germany). Constructs were introduced into Agrobacterium tumefaciens GV3101 strain by electroporation (Mersereau et al., 1990 (link)), and the A. thaliana reference line (Döring et al., 2019 (link)) was transformed following the floral dip method (Logemann et al., 2006 (link)). Plants were grown either at greenhouse conditions of 14 h light/day at a photon flux density (PFD) of ~300 μmol m−2 s−1 and at 21–22°C or in growth chambers operating at 16 h light/day (PFD: ~70 to 100 μmol m−2 s−1) and at a constant temperature of 21–22°C.
6
Cloning and Tagging Genes from Genomic DNA
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Genes amplified from gDNA were cloned into the pJET1.2 vector (CloneJET PCR Cloning Kit, Thermo Fisher Scientific) and clones were selected on ampicillin (100 mg/L). Plasmids were isolated and used as a template for a second PCR with primers designed for cloning into the pEHISTEV vector (NcoI/HindIII), in order to add the His-TEV tag at the N-terminus of the protein. Clones were selected on kanamycin (50 mg/L). Sequences were confirmed by Sanger sequencing (LGC Genomics).
7
Cloning System Gene Amplification
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To be compatible with the 1st generation cloning system, PCR primers were designed to amplify the genes lacking start and stop codons. All genes were amplified using ALLin HiFi DNA Polymerase (highQu) and the purified fragments were ligated into the EheI-digested, modified pFastBac1 plasmids using T4 DNA Ligase (Thermo Scientific) supplemented with 0.2 µL FastDigest EheI (Thermo Scientific) per 20 µL ligation volume. Ligated plasmids were amplified in E. coli DH5α cells (NEB C2987) and correctness of the insertion was verified by Sanger sequencing (LGC Genomics).
8
Kpn-Nhe Cloning of Genes
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To be compatible with the 2nd generation cloning system, PCR primers were designed to amplify the genes lacking start and stop codons while adding KpnI and NheI restriction sites at the 5’ and 3’ ends, respectively. Ligation of the digested PCR products into the equally restricted, modified pFastBac1 plasmids was performed using T4 DNA Ligase (Thermo Scientific). Ligated plasmids were amplified in E. coli DH5α cells (NEB C2987) and correctness of the insertion was verified by Sanger sequencing (LGC Genomics).
All primers used are listed in Supplementary Table3 .
All primers used are listed in Supplementary Table
9
Optimized Primer Design for Library Prep
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Primers were designed following a predesigned template (Supplemental Data 3 ), using PrimerBLAST (Ye et al, 2012 (link)). Potential primers were tested by using them individually in the first tailed-PCR of the targeted library preparation with either 3′ or 5′ test cDNA, with 30 cycles and a temperature gradient for the annealing temperature. Primers were initially evaluated by analyzing fragment sizes in the PCR product using Bioanalyzer (Agilent) or TapeStation (Agilent). If a fragment of the expected length was detected, the correct sequence was confirmed using Sanger sequencing (LGC Genomics). Primers for multiplex reactions were first optimized individually and then tested in multiplex in a similar setup.
10
Epidermal and Intestinal PGP-9 Expression
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Plasmids for transgenesis were assembled using the NEB HIFI DNA Assembly Kit (New England Biolabs Inc., Ipswich, MA, USA) according to the manufacturer’s instructions into the pUC19 vector linearised with SmaI (ThermoFisher, Waltham, MA, USA). Plasmid constructs were Cel-col-19p::Pun-pgp-9::FLAG::Cel-unc-54_3′-UTR utilising the col-19 promotor [39 (link)] to drive epidermal Pun-PGP-9 expression (Supplementary Figure S3a ) and Cel-ges-1p::Pun-pgp-9::FLAG::Cel-unc-54_3′-UTR (Supplementary Figure S3b ) utilising the ges-1 promotor [38 (link)] to drive intestine-specific Pun-PGP-9 expression. The C. elegans unc-54 3′-UTR [41 (link)] and the Pun-pgp-9 cDNA [23 (link)] were amplified from verified plasmids, while the 3′ end primer for the Pun-pgp-9 amplification introduced an in-frame FLAG-tag (DYKDDDDK) before the stop codon (all primers in Supplementary Table S3 ). The C. elegans promotors col-19p and ges-1p [38 (link)] were amplified from genomic DNA extracted from the Bristol N2 strain [39 (link)]. A co-injection marker plasmid (pPD118.33) driving pharyngeal GFP expression was used (Addgene L3790 plasmid #1596 was a gift from A. Fire). Sequences of all constructs were confirmed by Sanger-sequencing (LGC Genomics, Hoddesdon, UK).
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