Abi prism 3100 genetic analyser

Haplotyping was performed using the microsatellites D7S1824, D7S1826 up-stream of KCNH2 and D7S636, D7S3070, D7S483 and D7S1803 downstream of KCNH2 (Figure 1). CentiMorgan distances were obtained from the Map-O-Mat database for microsatellites (http://compgen.rutgers.edu/Mapomat/). PCR amplicons were generated using fluorescently end-labelled primers (available at NCBI UniSTS) at 0.4 μM per primer, per reaction. A loading mix of 0.5 μl amplicon, 9 μl HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 0.5 μl 600LIZ size standard (Applied Biosystems) was prepared, and DNA products were electrophoresed on an ABI PRISM® 3100 Genetic Analyser. Data were analysed using ABI GeneMapper software v4.0 (Applied Biosystems).

Published primers of the 5′-half of the VP1-coding region of the genome (primer 222 [5′-CICCIGGIGGIAYRWACAT-3′] and primer 224 [5′-GCIATGYTIGGIACICAYRT-3′]) were used for PCR amplification and sequencing^{53 (link)}. The 3C protease region was amplified with primer pair D1 [5′-TACAAACTGTTTGCTGGGCA-3′] and U2 [5′-TTCTTTTGATGGTCTCAT-3′]^{14 (link)}. Amplified products were purified using E.Z.N.A Cycle-Pure Kit (Omega Bio-tek. Inc.) according to the manufacturer’s instructions. Cycle sequencing reactions with both primers pair were performed by the ABI BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems). The ABI PRISM 3100 Genetic Analyser (Applied Biosystems) was used for electrophoresis and data collection. The sequences data were submitted to GenBank (accession numbers KC128616-KC128648, KC184859-KC184890, KC205656-KC205727, and KC286913-KC287081).

Frozen tissue samples available from thyroid tumours (n = 118) and adjacent/collateral normal thyroid tissues (n = 19) were crushed and homogenized. Total RNA from tissues and thyroid cancer derived cell lines was extracted using TRIzol® Reagent (Ambion®, Life Technologies™, CA, USA), according to the manufacturer’s protocol. Genomic DNA was extracted using the Genomic DNA Purification Kit (Citomed, Lisbon, Portugal) according to the manufacturer’s protocol.
cDNA was synthesized from 1 μg of RNA at 42 °C for 60 min, using oligo (dT) primers and M-MuLV reverse transcriptase (Fermentas, Thermo Scientific, St. Leon-Rot, Germany). The reverse-transcription PCR (RT-PCR) products were analysed by cDNA Sanger sequencing using the Big Dye terminator version 3.1 cycle (Applied Biosystems, Foster City, CA, USA). The samples were analysed in an automated sequencing machine (ABI Prism 3100 Genetic Analyser, Applied Biosystems, Foster City, CA, USA).

PCR amplifications were conducted using an Ex Taq PCR kit (TaKaRa, Kyoto, Japan) for three different molecular markers: cytochrome c oxidase subunit I (COI), 16S rRNA PageBreak(16S), and 18S rRNA (18S). Two oligonucleotide primers (0.2 µM each) and <1 µg of DNA template were added to the reaction mixtures. Thermal cycling was performed as follows: denaturing at 96 °C for 20 s, annealing at 55 °C for 45 s, and extension at 72 °C for 2 min for a total of 35 cycles. The oligonucleotide primer sequences used for the PCR amplification are shown in Table 1. The molecular sizes of the PCR products were confirmed with 1.2% Agarose S (Nippon Gene, Toyama, Japan) gel electrophoresis. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The DNA sequencing reaction was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Specific primers for each gene were used in sequencing reactions according to the manufacturer’s recommended procedure (Table 1). Sequencing was performed using an ABI PRISM 3100 genetic analyser (Applied Biosystems).

A DNA fingerprinting analysis was performed to demonstrate the origin of the established iPSC lines from the starting fibroblasts. The subsequent markers were amplified by PCR, followed by a fragment analysis: D13S317, D7S820, VWA, D8S1179, D21S11, D19S433, D2S1338, and amelogenin for sex determination. The analysis was carried out using the software ABI PRISM 3100 Genetic analyser and Peak Scanner v3.5 (Applied Biosystems, Waltham, MA, USA). The PCR primers can be consulted in Ortuño–Costela, et al., 2017 [28 (link)].

The full-length UL52 gene was amplified after DNA extraction (E-Mag, bioMérieux Craponne, France) from isolates by nested PCR using external and internal primers (Table S2). External PCR and internal PCR were both composed of 500ng of DNA (5µL of external PCR product for internal PCR), 25 µL of the 2X Prime START^® Max DNA polymerase (Takara, Maebashi, Japan), 0.3 nM of primers Ext1/Ext2 or Int1/Int2 (described in Table S2) qsp 50 µL H₂O PCR grade. The protocol was as follows: an initial denaturation for 5 min at 98 °C then a denaturation for 1 min at 98 °C; 10 s at 98 °C followed by 15 s at 55 °C and 2 min at 72 °C for 45 cycles; a final elongation step for 5 min at 72 °C. After purification of the PCR products using the Wizard^® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) according to the manufacturer’s instructions, internal PCR products were directly sequenced using the ABI Prism BigDye^® Terminator v3.1 Cycle Sequencing Kit in an ABI Prism 3100 Genetic Analyser (Applied Biosystems, Villebon-sur-Yvette, France) with nine sequencing primers designed using Geneious 9.1.8 software. Their specificity was assessed by comparison with GenBank^®. The sequencing results were analyzed using the Geneious 9.1.8 software by comparison with the AD169 UL52 sequence.