Quantiflour dsdna system

Calcium colorimetric assay (Catalog MAK022) was purchased from Sigma-Aldrich. Similar to previous studies, human ADSCs were seeded on our piezo- and non-piezo films, contrasting to cells seeding on tissue culture plates. Cells grew in respective media and underwent daily ultrasound treatments for one week. Next, media were removed, and cells were washed with Dulbecco’s PBS before lysed with 1ml of 2% v/v Triton X-100. Lysates were then quantified for intracellular calcium following manufacturer’s protocol. Meanwhile, relative cell numbers were quantified with Quantiflour dsDNA system (Catalog E2671, Promega).

Bisulfite conversion and purification of cell-free DNA samples were conducted using EZ DNA Methylation-Gold Kit (Zymo research, D5006, USA), following the manufacturer’s instructions. Subsequently, bisulfite-converted DNA samples were denatured to single stranded then processed with Adaptase™ technology adding adapters and unique dual indexes using xGen™ Methyl-Seq DNA Library Prep Kit (Integrated DNA Technologies, 10009824, USA). DNA concentrations were measured using the QuantiFlour dsDNA system (Promega, USA).

According to the manufacturer’s instructions, bisulfite conversion and cfDNA purification were prepared by EZ DNA Methylation-Gold Kit (Zymo Research, D5006, USA). DNA library was prepared from bisulfite-converted DNA samples using xGen Methyl-Seq DNA Library Prep Kit (Integrated DNA Technologies, 10009824, USA) with Adaptase technology, according to the manufacturer’s instructions. The QuantiFlour dsDNA system (Promega, USA) was used to analyze the concentration of DNA.

For individuals with T1D, an Omixon Holotype HLA V3 kit (Omixon, Hungary) was used on genomic DNA (0.8–1.2 µg) extracted by the QiAmp DNA blood mini kit, following the manufacturer’s protocols. The HLA typing kit generated DNA libraries and sequences for 11 loci, and among them were the DQA1, DQB1, and DRB1 genes. The protocol involved long-range PCR amplification of HLA genes using locus-specific master mixes, followed by quantitation and normalization of the resulting PCR amplicon, using QuantiFlour dsDNA system (Promega, USA). Amplicons were then subjected to enzymatic fragmentation, were end repaired and adenylated, followed by index ligation. The resulting single pool of indexed libraries were selected using AMPure XP magnetic beads (Beckman Coulter, USA) and were quantified using the qubit fluorometer (Thermofisher Scientific, USA). Next-generation sequencing (NGS) was carried out on an Illumina Miseq (Illumina, USA) sequencer, following the manufacturer’s protocols.