EZ DNA Methylation kit

DNA methylation profiling Gene specific profiling - HEK293T RBP3

Experiment
DNA methylation profiling Gene specific profiling - HEK293T RBP3
Product
EZ DNA Methylation kit from Zymo Research
Manufacturer
Zymo Research

Protocol tips

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
Protocol tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

Bisulfite Sequencing
Bisulfite modification of genomic DNA was performed using either the Epitech (Qiagen, Valencia, CA) or EZ DNA Methylation (Zymo, Irvine, CA) kits. MethPrimer was used to design primers to specifically amplify photoreceptor specific genes and the putative promoter regions of two intergenic miRNAs ( and ) (, , ). For direct sequencing, the PCR products were agarose gel-purified and directly sequence by the High-Throughput Genomics Unit at the University of Washington using the forward primers. Using the ContigExpress function of Vector NTI Advance 10 (Invitrogen, Carlsbad, CA), the chromatograph peak heights for C and T at each CpG site were measured and the % methylation (C peak height/(T peak height+C peak height)) versus CpG position, with respect to the TSS, was plotted . For sequencing of individual clones after PCR amplification, the presence of a specific PCR product was confirmed by agarose gel electrophoresis, and the PCR product for each set of primers was subcloned using the TOPO-TA cloning kit (Invitrogen, Valencia, CA). Thirty sequences (10 clones for each of 3 biological replicates) with ≥95% conversion of the non-CpG cytosines were obtained and sequenced using the M13 reverse primer by the University of Washington High-throughput Genomics Unit. Fasta files of each sequence were analyzed using a custom JAVA program (DNAMethylMap, unpublished software by B. Kumar and S. Yegnasubramanian).

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Reviews

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Discussion

Discussion

2 years ago

Author: Jody Hancock Canada

How can I preserve leukocytes for DNA methylation profiling?

I would like to preserve leukocytes for future epigenetic analysis. How can I preserve them effectively in order to perform DNA methylation profiling at a later time?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - HEK293T RBP3 using EZ DNA Methylation kit from Zymo Research.

Paper title
Cell-Specific DNA Methylation Patterns of Retina-Specific Genes
Full paper
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Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation kit below.

Download PDF Download manufacturer protocol

Videos

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