5200 fragment analyzer instrument

Primer pairs for the amplification of all ten exons of the NSDHL gene were described previously [8 (link)] and are given in Table S2. PCR products for each exon were amplified from genomic DNA using AmpliTaq Gold 360 Master mix (Thermo Fisher Scientific, Reinach, Switzerland). PCR products were visualized using a 5200 Fragment Analyzer instrument (Agilent, Basel, Switzerland), which uses capillary electrophoresis to enable accurate sizing and quantification of nucleic acids. All exons of the NSDHL gene were analyzed by direct Sanger sequencing of PCR amplicons. After treatment with exonuclease I and alkaline phosphatase, amplicons were sequenced on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific, Reinach, Switzerland). Sanger sequences were analyzed using the Sequencher 5.1 software (Gene Codes, Ann Arbor, MI, USA). For the RT-PCR on skin cDNA, a forward primer located at the boundary of exons 6 and 7 together with a reverse primer located in exon 10 was used (Table S2). After an initial denaturation of 10 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 60 s at 72 °C were performed, followed by a final extension step of 7 min at 72 °C. The RT-PCR products were analyzed on a Fragment Analyzer and sequenced as described above.

DNA libraries were prepared from extracted gDNA using the NEBNext Ultra II FS DNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s instructions with some modifications. Adaptors ligation was performed using preannealed oSh-F and oSh-R primers (Table S2) at a working concentration of 2 μM. Size selection and cleanup of adaptor-ligated DNA were done using AMPure XP beads (Beckman Coulter). Libraries were amplified by quantitative PCR using Veraseq 2.0 high-fidelity DNA polymerase (Enzymatics) and Illumina i5 and i7 index primers with the following protocol: (i) 30 sec at 98°C, (ii) 35 cycles of 15 sec at 98°C, 30 sec at 60°C, and 18 sec at 72°C, and (iii) 2 min at 72°C. Reactions were stopped before reaching the amplification plateau. Library quality was assessed on a 5200 fragment analyzer instrument (Agilent) and DNA concentration was measured using the Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher). A total of 15 different libraries were prepared, in 3 biological replicates. Illumina sequencing was performed by the McGill Genome Center using an Illumina NovaSeq instrument.

DNA libraries were prepared from extracted gDNA using the NEBNext Ultra II FS DNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s instructions with some modifications. Adaptors ligation was performed using preannealed oSh-F and oSh-R primers (Table S2) at a working concentration of 2 μM. Size selection and cleanup of adaptor-ligated DNA were done using AMPure XP beads (Beckman Coulter). Libraries were amplified by quantitative PCR using Veraseq 2.0 high-fidelity DNA polymerase (Enzymatics) and Illumina i5 and i7 index primers with the following protocol: (i) 30 sec at 98°C, (ii) 35 cycles of 15 sec at 98°C, 30 sec at 60°C, and 18 sec at 72°C, and (iii) 2 min at 72°C. Reactions were stopped before reaching the amplification plateau. Library quality was assessed on a 5200 fragment analyzer instrument (Agilent) and DNA concentration was measured using the Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher). A total of 15 different libraries were prepared, in 3 biological replicates. Illumina sequencing was performed by the McGill Genome Center using an Illumina NovaSeq instrument.