Genomic DNA was extracted from ccRCC and pair-matched adjacent normal tissues, and the bisulfite conversion reaction was performed according to the manufacturer’s instructions. The PCR mixtures were pre-heated for 4 min at 94 °C, followed by 45 cycles of 94 °C for 20 s, 56 °C for 30 s and 72 °C for 1 min, the final extension at 72 °C for 3 min. PCR products were incubated with Shrimp Alkaline Phosphatase following the manufacturer’s protocol. After in vitro transcription and RNase A digestion, small RNA fragments with CpG sites were acquired for the reverse reaction. The methylation ratios of the products were calculated using Epityper software Version 1.0 (Sequenom, San Diego, CA, USA). The Sequenom MassARRAY platform (Oebiotech, Shanghai, China) was utilized to quantitatively analyze the DNA methylation status of ZNF582-AS1 DNA. PCR primers were designed using EpiDesigner, and their sequences were listed in Additional file 1 : Table S1.
Epityper software version 1
Manufactured by Labcorp
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The Epityper software version 1.0 is a tool designed for data analysis and processing. Its core function is to facilitate the interpretation and management of raw data generated from various analytical instruments.
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Lab products found in correlation
Massarray platform, by Labcorp (13 mentions)
Sequenom massarray platform, by CapitalBio (6 mentions)
Massarray compact maldi tof, by Labcorp (4 mentions)
Ez dna methylation gold kit, by Zymo Research (4 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
Massarray platform, by CapitalBio (3 mentions)
Sequenom massarray platform, by OE Biotech (2 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Spectrochip, by Labcorp (2 mentions)
Epityper assay, by Labcorp (1 mentions)
Massarray, by Labcorp (1 mentions)
Epidesigner, by Labcorp (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Masscleave kit, by Labcorp (1 mentions)
Massarray compact system, by Labcorp (1 mentions)
Ez 96 dna methylation kit, by Zymo Research (1 mentions)
Epidesigner software, by Labcorp (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Mastercycler gradient, by Eppendorf (1 mentions)
27 protocols using epityper software version 1
1
Quantitative Analysis of ZNF582-AS1 DNA Methylation
2
Quantitative Methylation Analysis of Imprinted Genes
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As the previously published method [16 (link)], the Sequenom MassARRAY platform (CapitalBio, Beijing, China) was used to perform the quantitative methylation analysis of the imprinted genes. This system uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). A detectable pattern was then analyzed for its methylation status. The spectral methylation ratios were generated using Epityper software version 1.0 (Sequenom, San Diego, CA, USA).
3
Quantitative Methylation Analysis of ALDH2 Promoter
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The Sequenom MassARRAY platform was used for the quantitative methylation analysis of the upstream sequence of ALDH2 gene promoter. The methylation status of a detected pattern was then analyzed using Epityper software version 1.0 (Sequenom, San Diego, CA, USA). The promoter regions of the upstream sequence were chosen according to the website: http://genome.ucsc.edu . PCR primers used in this system (Table 2 ) were designed using predict software (Methyl Primer Express v1.0.exe).
The procedures and reaction system were reported before [15 (link)]. The region analyzed and the CpG sites of the upstream sequence are shown inFigure 2 and Table 3 . The same experiments were repeated in triplicate. The methylation level was presented as the ratio of methylated cytosines over the total number of methylated and unmethylated cytosines.
The procedures and reaction system were reported before [15 (link)]. The region analyzed and the CpG sites of the upstream sequence are shown in
4
Quantitative Methylation Analysis of EFEMP1
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Genomic DNA was extracted from the hippocampus with the QIAamp DNA Mini Kit (Qiagen 51304, Germany) according to the manufacturer's instructions. The concentration and purity of the DNA were determined by absorbance at 260 and 280 nm. A total of 1.5 μg genomic DNA from each sample was bisulfite-treated with the EZ DNA Methylation-Gold Kit (Cat: D5005, Zymo Research, USA). The Sequenom MassARRAY platform (CapitalBio, Beijing, China) [19 (link)] was used to perform the quantitative methylation analysis of EFEMP1 (GenBank accession number NC_005113). A detectable pattern is then analyzed for its methylation status. PCR primers were designed with EpiDesigner (http://www.epidesigner.com ). For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10-mer tag on the forward primer to adjust for melting temperature differences. We used the following primers on the basis of the reverse complemented strand of EFEMP1: 5′-aggaagagagTGTTTAATTTAATTTTGGATGGGTAG-3′and 3′-cagtaatacgactcactatagggagaaggctAAAAATAAACCAAATAAAACTCATCCT-5′. Mass spectra were obtained via MassARRAY Compact MALDI-TOF (Sequenom, San Diego, CA), and their methylation ratios (n = 4, each group) were generated using the EpiTYPER software version 1.0 (Sequenom, San Diego, CA).
5
DNA Methylation Analysis Workflow
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The genomic DNA of tissue or peripheral blood was extracted using the phenol chloroform method. DNA concentration, OD260, OD280, and OD260/OD280 were measured using a nanodrop 1000 spectrophotometer. The DNA sample OD260/OD280 used in this experiment was 1.8–1.9, which was regarded as qualified DNA. The DNA samples were treated with bisulfite, amplified by PCR, and reacted with a self-analysis platform. The 384-pore plate was put into the sample adding instrument. After the parameter setting was complete, the sample could be spotted on the chip. After sampling, the chip was placed in the mass spectrometer, and methylation analysis was performed according to the molecular weight difference of C and T bases in the fragment. The methylation ratio of mass spectrometry was obtained by EpiTYPER software version 1.0 (Sequenom, San Diego, CA, United States).
6
Quantitative Analysis of hsa-let-7g Methylation
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The Sequenom MassARRAY platform (CapitalBio, Beijing, China) was used to perform quantitative analysis of the methylation of hsa‐let‐7 g. This system uses matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry in combination with RNA base‐specific cleavage (MassCLEAVE). The detected pattern is then analysed for its methylation status. PCR primers were designed with Methprimer (http://epidesigner.com ). For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, as well as a 10‐mer tag on the forward primer to adjust for melting temperature differences. Two pairs of primers were used to amplify the promoter region of hsa‐let‐7 g: L1: aggaagagagATTTGGGAGGTTGAGGTAAGAGTAT; R1: cagtaatacgactcactatagggagaaggctCAAAATTTCACCATATTAACCAAAA; L2: aggaagagagTATTTAGGGAGGTTTAGGAGAGTGG; and R2: cagtaatacgactcactatagggagaaggctACCTCCAAAACTCAAACAATCCT. The location of primers is shown in Figure 1 . In total, six CpG sites (which were divided into five CpG units) were examined in this region (shown in Fig. 1 ). The spectral methylation ratios were generated using Epityper software version 1.0 (Sequenom, San Diego, CA, USA).
7
Quantitative Analysis of HOXA1 Methylation
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Genomic DNA was extracted from the cells with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), and the bisulfite conversion reaction was performed using an EpiTect Bisulfite kit (QIAGEN) according to the manufacturer’s instructions. The Sequenom MassARRAY platform (Oebiotech, Shanghai, China) was utilized to quantitatively analyze the methylation status of the HOXA1 gene promoter. PCR primers (Additional file 6 : Table S5) were designed using EpiDesigner. The PCR mixtures were pre-heated for 4 min at 94 °C, followed by 45 cycles of 94 °C for 20 s, 56 °C for 30 s and 72 °C for 60 s, the final extension at 72 °C for 3 min. PCR products were incubated with Shrimp Alkaline Phosphatase following the manufacturer’s protocol. After in vitro transcription and RNaseA digestion, small RNA fragments with CpG sites were acquired for the reverse reaction. The methylation ratios of the products were calculated using Epityper software Version 1.0 (Sequenom, San Diego, CA, USA).
8
TERT Promoter Methylation Analysis
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The Sequenom MassARRAY platform was used for the quantitative methylation analysis of TERT promoter region. The methylation status of a detected pattern was then analyzed using Epityper software version 1.0 (Sequenom Inc, San Diego, CA, USA). Polymerase chain reaction (PCR) primers were as follows: forward: 5′-GAGTTTGGATTTTTGGGAAGTTT-3′, reverse: 5′-TAAAACCAACATCTAATCACATCCC-3′. The amplified regions used for Sequenom are shown in Figure 1 . The quantitative results were referred to as cytosine-phosphate-guanine (CpG) units (units containing either one individual CpG site or multiple consecutive CpG sites). Methylation levels were presented as the ratio of methylated cytosines over the total number of methylated and unmethylated cytosines. TERT methylation was calculated as the mean of all CpG units. The cutoff of TERT methylation in tumor tissues was set at 35.5% according to a receiver operating characteristic (ROC) analysis having balanced sensitivity and specificity. TERT methylation greater or equal to 35.5% was considered as hypermethylated.
The CpG units that produced data for less than 30% of samples (unreliable CpG units) as well as samples lacking more than 30% of their data points (unreliable samples) were discarded.18 (link)
The CpG units that produced data for less than 30% of samples (unreliable CpG units) as well as samples lacking more than 30% of their data points (unreliable samples) were discarded.18 (link)
9
Quantifying DNA Methylation in GNAS Cluster
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Three DMRs have been identified in the GNAS cluster: an extensive germline maternally methylated region at the Nespas promoter; a maternally methylated germline region at the Exon1A promoter; and a paternally methylated region spanning the Nesp promoter. DNA methylation at the indicated loci was measured using the Sequenom MassARRAY platform (CapitalBio, Beijing, China). This system uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). A detectable pattern was then analyzed for its methylation status. Polymerase chain reaction (PCR) primers were designed using Methprimer (http://epidesigner.com ). The PCR primers specific for bisulfate-converted DNA for the DMRs in the GNAS imprinting cluster are listed in Supplementary Table 2 . The locations of the amplicons used for methylation analysis are shown by the short bars under each DMR (Figure 2 ). The methylation ratios of the spectra were generated using Epityper software version 1.0 (Sequenom, San Diego, CA, USA).
10
Quantitative Methylation Analysis of OPRM1 Promoter
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