Bigdye terminator v1.1 sequencing kit

Confirmation of the NGS data concerning the COL5A1 candidate variant was first performed by Sanger sequencing on DNA extracted from the proband’s and her parents’ peripheral blood. Subsequently, Sanger sequencing was also carried out on DNA extracted from proband’s and her father’s hair bulbs, nails and saliva. For Sanger sequencing on DNA extracted from hair bulbs, nails and saliva, the experiment also included a control DNA extracted from the same tissues. The primers were designed by using primer3 tool (https://primer3.ut.ee/, accessed on 26 July 2021) to amplify COL5A1 exon 9, checked by BLAST against the human genome to ensure its specificity (COL5A1_ex9_F: 5′-cctcctcgtctgaaggtgataa-3′, COL5A1_ex9_R: 5′-tgacttttccacaattctctagct-3′). The amplified products were subsequently purified by using ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher Scientific, Wilmington, DE, USA) and sequenced by using a BigDye Terminator v1.1 sequencing kit (Thermo Fisher Scientific, Wilmington, DE, USA). The fragments obtained were purified using DyeEx plates (Qiagen, Tübingen, Germany) and resolved on an ABI Prism 3130 Genetic Analyzer (Thermo Fisher Scientific, Wilmington, DE, USA). Sequences were analyzed using Sequencer software (Gene Codes, Ann Arbor, MI, USA). The identified variant was resequenced in independent experiments.

Fragments of 1011bp, 1156bp, and 968bp encompassing the miR-200c-3p binding site located in the 3′-UTR of SULF1, PRKG1, and SIDE genes, respectively, were amplified by using Taq polymerase Pfu (Promega, Madison, WI, USA). Primers for PCR were designed according to the 3’-UTR genomic sequence of human SULF1, PRKG1, and SIDE genes (Table S1). After PCR amplification, PCR products were purified and subjected to restriction enzyme digestion with XhoI (New England BioLabs, Ipswich, MA, USA) and XbaI (New England BioLabs, Ipswich, MA, USA) and then cloned into the pmir-GLO dual-luciferase miRNA target expression reporter vector (Promega, Madison, WI, USA).
In vitro mutagenesis was employed to delete the miR-200c-3p binding site from pmir-SULF1, pmir-PRKG1, and pmir-SIDE by using the QuickChange II site-directed mutagenesis kit (Stratagene, Santa Clara, CA, USA) according to the manufacturer’s instructions. All constructs containing wild-type and mutated fragments were verified by Sanger sequencing using BigDye Terminator v1.1 sequencing kit (Thermo Fisher Scientific, Waltham, MA, USA), purified using DyeEx plates (Qiagen, Hilden, Germany), and resolved on ABI Prism 3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Sequences were analyzed using the Sequencer software (Gene Codes, Ann Arbor, MI, USA).

Genomic DNA (gDNA) was extracted from FFPE using the Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech (Shanghai) Co., Ltd.). gDNA was amplified with the forward and reverse primers. After purified PCR products were detected by agarose gel electrophoresis (150 V, 100 mA, 10–20 min), DNA was extracted from the agarose gel by a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech (Shanghai) Co., Ltd.), and then Sanger-sequenced with the BigDye terminator v1.1 sequencing kit and a 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). The results were analyzed by Variant reporter software version 2.1 (Applied Biosystems).

A subset of 20 samples was randomly selected for sequencing to confirm the PCR results. Cag28F and cag4 primers were used to amplify the variable region and generate ~650 to ~850-bp amplicons. The PCR reaction was conducted in a 50-μl volume with 15 pmol of each primer, 0.3 mM dNTPs, 2 mM MgCl₂, and 1 U of Platinum® Taq DNA Polymerase (Invitrogen Carlsbad, CA, USA) per reaction. The amplification conditions were as follows: 1 cycle at 94°C for 5 min; 30 cycles at 94°C for 40 s, 55.5°C for 30 s, and 72°C for 50 s; and a final extension cycle at 72°C for 7 min. The PCR products were purified with the PureLink® PCR Purification Kit (Invitrogen Carlsbad, CA, USA) according to the manufacturer’s instructions. The purified products were sequenced with the BigDye terminator v1.1 sequencing kit (Applied Biosystems, Foster City, CA, USA) and analyzed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The nucleotide sequences were transformed into amino acid sequences with MEGA v5 software [43 (link)]. The ClustalW option within the MEGA software was used to generate a multiple amino acid sequence alignment. The partial CagA protein sequence from the H. pylori strain 43526 (GenBank: AF001357.1) was used as a reference.