Epidesigner software

Quantitative analysis of differentially methylated regions was performed by BioMiao Biological Technology (Beijing, China) with the Agena MassARRAY platform (Agena, San Diego, USA). Briefly, genomic DNA was isolated from HL-7702 cell lines, and 2 μg DNA was treated with sodium bisulfite using an EZ DNA Methylation-Gold kit (ZYMO Research) according to the manufacturer’s instructions. The specific primers based on the reverse complementary strands of the
TFEB promoter were designed using EpiDesigner software (Agena), and the quantitative results for each CpG or multiple CpGs were analyzed with EpiTYPER
^TM (Agena). The primer sequences were 5′-aggaagagagAGGTATTTAAGGGTATTTTTGGTGG-3′ and 3′-cagtaatacgactcactatagggagaaggctCCTATAATCCCAACATTTTAAAAAACC-5′. Bisulfite-modified DNA PCR amplifications were performed with a precycling hold at 94°C for 4 min and subjected to 45 cycles of 94°C for 20 s, 56°C for 20 s,72°C for 1 min, and a final extension at 72°C for 3 min. Further experimental analysis of the contents of DNA methylation was determined, as described previously
[18] (link).

The potentially bladder specific target region deduced from the comparison of the screening experiments in cell culture and urine was assessed in more detail by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry (MassARRAY EpiTYPER system, Agena Bioscience GmbH, Hamburg, Germany) which enables the quantitative measurement of CpG methylation at single dinucleotide resolution [22 (link),23 (link)]. For this purpose, primers covering a DNA-stretch which includes the two most promising candidate sites were designed by Agena`s EpiDesigner software (http://www.epidesigner.com/index.html). The sequences were aggaagagagGGGTTATGTTGAGAAGTAAGGAATGT (forward-primer) and cagtaatacgactcactatagggagaaggctCCCACACAAAACTTAAAAATAAAACTT (reverse primer, the small print represents the respective tags required by the method). The amplified region was CHR6:28911328-28911620 (genome build GRCh37/hg19). All analyses were carried out according to the protocol of Agena Bioscience GmbH and have been previously described in detail [15 (link)].