For standard controls, we used Human methylated and non-methylated DNA set from Zymo (Zymo, CA, USA). The non-methylated DNA was purified from HCT116 DKO cells knockout for both DNA methyltransferases DNMT1 (–/–) and DNMT3b (–/–). The methylated DNA was purified from the same HCT116 DKO cells and was enzymatically methylated by M.SssI methyltransferase. Five actual methylation percentage (0%, 25%, 50%, 75% and 100%) were prepared mixing different ratios of non-methylated and methylated human control DNA (Zymo, CA, USA) bisulphite converted (MethylEdge Bisulfite Conversion System, Promega). Additionally, we collected DNA from 18 potential BWS patients and 15 healthy controls. Genomic DNA was extracted from peripheral blood using Gentra Puregene Blood Kit (Qiagen) and DNA quality was determined by Qubit 2.0 (Invitrogen, ThermoFisher). Appropriate human subject approvals and written inform consent were obtained from all participants. Bisulphite conversion of genomic DNA was performed in all samples at the same time with MethylEdge Bisulfite Conversion System from Promega.
Methyledge bisulfite conversion system
Manufactured by Promega
Sourced in United States
The MethylEdge Bisulfite Conversion System is a laboratory tool designed for the treatment of DNA samples with sodium bisulfite. This process is a critical step in the analysis of DNA methylation patterns, a key epigenetic modification.
Automatically generated - may contain errors
Lab products found in correlation
High pure pcr template preparation kit, by Roche (2 mentions)
N1301, by Promega (2 mentions)
Methyledge bisulfite conversion system kit, by Promega (2 mentions)
D1521, by Promega (2 mentions)
Viia7 instrument, by Thermo Fisher Scientific (1 mentions)
Sybr green rt pcr master mix, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Promega (1 mentions)
Hotstartaq, by Qiagen (1 mentions)
Pureyield plasmid miniprep system, by Promega (1 mentions)
Top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Zymotaq premix, by Zymo Research (1 mentions)
Takara epitaq hs, by Takara Bio (1 mentions)
Methylated human control, by Promega (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Human methylated and non methylated dna set, by Zymo Research (1 mentions)
Gentra puregene blood kit, by Qiagen (1 mentions)
Pyromark q24, by Qiagen (1 mentions)
Quantifiler human dna quantification kit, by Thermo Fisher Scientific (1 mentions)
Snapshot multiplex kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using methyledge bisulfite conversion system
1
Methylation Profiling of Beckwith-Wiedemann Syndrome
2
Bisulfite Sequencing of hTERT Promoter
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Genomic DNA was treated with bisulphite using the MethylEdge™ Bisulfite conversion system (Promega, United States) in accordance with the manufacturer’s instructions. Thirty nine CpG sites of the hTERT promoter, extending 500 bases upstream of the transcriptional start site to 500 bases downstream of the translational start site, were examined for methylation. The primers targeting promoter region of hTERT gene for amplifying bisulphite-modified DNA were: forward primer 5’-CTACCCCTTCACCTTCCAA-3′, and reverse primer 5′-GTTAGTTTTGGGGTTTTAGG-3′. Thermocycling conditions were an initial step at 95°C (5 min), then 45 cycles of the following steps: 95°C (60 s), 59°C (30 s) and 72°C (35 s). 3 μL of the PCR product was visualized on a 1.5% agarose gel. Each PCR product was sequenced using the hTERT forward primer on SeqStudio™ Genetic Analyzer System with SmartStart, (Thermofisher, United States). Sequence reads were visualized by Bio Edit Sequence Alignment Editor software (Alzohairy, 2011 ). Sequences with noisy data background were trimmed and introduced to the BiQ analyzer software which displays the methylated sites and unmethylated sites separately (Akgün et al., 2022 (link)).
3
CDKN2A Promoter Methylation Analysis
4
Genomic DNA Extraction and Bisulfite Conversion
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Genomic DNA was isolated from frozen colon tissues using the High Pure PCR Template Preparation Kit (Roche) according to the manufacturer’s protocol. DNA concentration and quality were measured using the NanoDrop ND-1000 spectrophotometer. Genomic DNA was modified by sodium bisulfite to convert unmethylated cytosines to uracil using the available MethylEdge™ Bisulfite Conversion System (Promega, Madison, WI) as per manufacturer’s instructions. Bisulfite-modified DNA specimens were aliquoted and stored at −20°C.
5
DNA Methylation Analysis of Fish Oocytes
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Unfertilized oocytes were squeezed out from anesthetized adult female fish. Oocytes were activated in egg water for 10 min. Eggs that failed to activate or had lysed were discarded. Healthy eggs were collected into tubes and lysed in a chilled cell lysis solution (10 mM Tris pH8, 10 mM NaCl, 0.5% NP40) by passing them through a 20 G needle. Nuclei were collected by centrifugation at 3500 g for 5 min at 4 °C. The supernatant was removed and the nuclei pellet lysed in nuclear lysis buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS, 10 μl Proteinase K, 2 μl RNase A/ml) at 55 °C for 2 h. DNA was extracted by phenol chloroform extraction. DNA was digested again with RNase A at 37 °C for 3 h, followed by phenol chloroform extraction. DNA from 500 eggs was subjected to bisulfite conversion using MethylEdge Bisulfite Conversion System (Promega). PCR was performed with HotStarTaq (Qiagen) and PCR product TA-cloned into pGEM-T Easy (Promega). 16 clones were picked per genotype and sequenced with T7 primer. Efficiency of bisulfite conversion was over 99% as observed by the conversion of non-CpG cytosines.
6
Bisulfite Sequencing of FCGRT Promoter
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DNA from cells was extracted using the E.Z.N.A. Tissue DNA kit and converted with bisulfite using the MethylEdge Bisulfite Conversion System (Promega). Amplicons corresponding to the FCGRT 5′UTR were amplified using ZymoTaq PreMix (Zymo Research) and specific primers (Table S2 ). Amplicons were cloned into the pCR4-TOPO vector and transformed into TOP10 chemically competent E. Coli following the manufacturer’s instructions (Thermo Fisher Sci.). Individual clones (n = 4-8) for each amplicon were selected, and grown in LB broth supplemented with ampicillin (Sigma-Aldrich). Plasmid DNA was extracted from each clone using the PureYield Plasmid Miniprep System (Promega) and directly sequenced at the Genomics Shared Resource at the Roswell Park Comprehensive Cancer Center (Buffalo, New York).
7
Bisulfite Conversion via Droplet-based Method
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Barcoded DNA in 20 μl low-EDTA TE buffer was heated at 95 °C for 1.5 min and immediately put on ice for 2 min to produce single-stranded DNA. The DNA solution was then mixed with 130 μl bisulfite conversion reagent (Promega, N1301, MethylEdge® Bisulfite Conversion System) before being loaded into the bisulfite droplet device (Supplementary Fig. 2 ) at a flow rate of 5 μl/min, with the oil’s flow rate at 15 μl/min. The collected bisulfite droplets were incubated at 95 °C for 5 min and 54 °C for 1 h for bisulfite conversion in droplets. Droplets were then broken to separate the aqueous phase into a new tube. DNA desulphonation and cleanup steps were immediately performed following the manufacturer instructions of the MethylEdge® Bisulfite Conversion System kit from Promega. After the column cleanup, DNA was selected by 1.1X SPRIselect bead and eluted into 11 μl low-EDTA TE buffer. We examined the bisulfite conversion rate using unmethylated lambda phage DNA (Promega, D1521).
8
Methylation-Specific PCR for Tumor Biomarkers
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After isolation from tumor tissue, DNA was treated with bisulfite using the MethylEdge Bisulfite Conversion System (Promega, Madison, WI, USA) following the manufacturer’s instructions. Bisulfite-treated DNA was afterward used for methylation-specific PCR (MSP).
Primer sequences of DKK1, DKK3, and GSK3β promoter region for MSP were synthesized according to [31 (link),32 (link),33 (link)], respectively (Table 1 ).
PCRs for bisulfite-treated DNA were performed using TaKaRa EpiTaq HS (TaKaRa Bio, USA): 1XEpiTaq PCR Buffer (Mg2+ free), 2.5 mM MgCl2, 0.3 mM dNTPs, 20 pmol of each primer (Sigma-Aldrich, USA), 50 ng of DNA, and 1.5 units of TaKaRa EpiTaq HS DNA Polymerase in a 25 µL final reaction volume. PCR cycling conditions are shown inTable 2 .
PCR products were separated on 2% agarose gel stained with Syber Safe nucleic acid stain (Invitrogen, Thermo Scientific, Waltham, MA, USA) and visualized on a UV transilluminator. Methylated Human Control (Promega, Madison, WI, USA) was used as a positive control for the methylated reaction, while unmethylated DNA EpiTect Control DNA (Qiagen, Hilden, Germany) served as a positive control for the unmethylated reaction.
Primer sequences of DKK1, DKK3, and GSK3β promoter region for MSP were synthesized according to [31 (link),32 (link),33 (link)], respectively (
PCRs for bisulfite-treated DNA were performed using TaKaRa EpiTaq HS (TaKaRa Bio, USA): 1XEpiTaq PCR Buffer (Mg2+ free), 2.5 mM MgCl2, 0.3 mM dNTPs, 20 pmol of each primer (Sigma-Aldrich, USA), 50 ng of DNA, and 1.5 units of TaKaRa EpiTaq HS DNA Polymerase in a 25 µL final reaction volume. PCR cycling conditions are shown in
PCR products were separated on 2% agarose gel stained with Syber Safe nucleic acid stain (Invitrogen, Thermo Scientific, Waltham, MA, USA) and visualized on a UV transilluminator. Methylated Human Control (Promega, Madison, WI, USA) was used as a positive control for the methylated reaction, while unmethylated DNA EpiTect Control DNA (Qiagen, Hilden, Germany) served as a positive control for the unmethylated reaction.
9
Bisulfite Conversion and Pyrosequencing
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Genomic DNA was bisulfite converted using the MethylEdge Bisulfite Conversion System (Promega, Madison, USA) following the manufacturer’s protocol. The region of interest was amplified by PCR and methylation level was measured by pyrosequencing using PyromarkQ24 (Qiagen France, Courtaboeuf, France). (Primers are listed in Online Supplementary Table S1).
10
Bisulfite Conversion of Barcoded DNA
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Barcoded DNA in 20 μl low-EDTA TE buffer was heated at 95 °C for 1.5 min and immediately put on ice for 2 min to produce single-stranded DNA. The DNA solution was then mixed with 130 μl bisulfite conversion reagent (Promega, N1301, MethylEdge® Bisulfite Conversion System) before being loaded into the bisulfite droplet device (Supplementary Fig. 2 ) at a flow rate of 5 μl/min, with the oil’s flow rate at 15 μl/min. The collected bisulfite droplets were incubated at 95 °C for 5 min and 54 °C for 1h for bisulfite conversion in droplets. Droplets were then broken to separate the aqueous phase into a new tube. DNA desulphonation and cleanup steps were immediately performed following the manufacturer instructions of the MethylEdge® Bisulfite Conversion System kit from Promega. After the column cleanup, DNA was selected by 1.1X SPRIselect bead and eluted into 11 μl low-EDTA TE buffer. We examined the bisulfite conversion rate using unmethylated lambda phage DNA (Promega, D1521).
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