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5mC Tet1 Oxidation Kit

Details for Product No. ABIN1741993, Supplier: Log in to see
Application
DNA Sequencing (Seq), Tet-assisted Bisulfite Sequencing (TAB-Seq)
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Purpose 5mC Tet1 Oxidation Kit can be used to convert 5-methylcytosine (5mC) in genomic DNA to 5-carboxylcytosine (5caC) for subsequent sequencing applications.
Characteristics Each kit contains enough reagents for up to 3 genome-wide or 9 loci-specific sequencing reactions based on recommended protocols.
Components
  • Tet1 oxidation reagent 1: 50 μL
  • Tet1 oxidation reagent 2: 180 μL
  • Tet1 protein: 50 μL
  • Proteinase K (20 mg/mL): 15 μL
Material not included
  • 5mC control DNA and primers
  • Micro Bio-Spin 30 Columns (BioRad)
  • QIAquick PCR Purification Kit (Qiagen)
  • EpiTect Bisulfite Kit (Qiagen) or MethylCode Bisulfite Conversion Kit (Invitrogen)
  • Zero Blunt TOPO PCR Cloning Kit (Invitrogen)
  • PfuTurbo Cx Hotstart DNA Polymerase (Agilent)
Protocol Important notes before starting
  • Store Tet1 at -80 °C or lower. Upon first use, aliquot Tet1 into single use portions. Avoid repeated freezing and thawing (up to three times). Multiple freeze/thaw cycles may result in decreased performance.
  • Store Tet1 oxidation reagents 1 and 2 at -80 °C. Upon first use, it is recommended to aliquot into single use portions.
  • The reaction solution should turn from transparent to slightly brown after mixing Tet oxidation reagents 1 and 2.
Reagent Preparation

For each 1 μg genomic DNA, add 5 ng (0.5 %) 5mC control DNA and sonicate the mixed genomic DNA to average 400 bp.

Assay Procedure

Tet1-based 5mC oxidation
1) Prepare the Tet1-based oxidation reaction. Add each component in the order listed below:

  • Milli-Q water: to 50 µL
  • Genomic DNA: 500 ng, up to 27 µL (final concentration: 10 ng/µL)
  • Tet oxidation reagent 2: 15 µL
  • Tet oxidation reagent 1: 3.5 µL
  • Tet1 protein: 5.0 µl
  • Final volume: 50 µL

For each 500 ng DNA to start with in Tet1 oxidation reaction, 200-300 ng of DNA can be recovered after purification (Step 4).
Note: If different amount of genomic DNA is applied, change the volume of the reaction accordingly (from 20 μL to 50 μL) with the same final concentration of all reagents.
Note: If there is more than 3 % of 5mC in the genomic DNA, increase the concentration of Tet1 accordingly.
2) Mix well and allow the reaction to proceed at 37 °C for 1 h.
3) Add 1 μL Proteinase K (20 mg/mL) to the reaction mixture and incubate at 50 °C for 1 h.
4) Purify the oxidized DNA with Micro Bio-Spin 30 Columns (Bio-Rad) first and then apply to QIAquick PCR Purification Kit (Qiagen). Elute DNA in 50 μL water.

Test conversion efficiency of 5mC to 5caC of the 5mC spike-in control DNA
5) Apply 50 ng Tet1-oxidized DNA from Step 4 to EpiTect Bisulfite Kit (Qiagen) or MethylCode Bisulfite Conversion Kit (Invitrogen) following the manufacture’s instruction.
6) Use the table below as guidance to prepare 50 μL PCR:
  • 10×PfuTurbo Cx reaction buffer: 5 µL (final concentration: 1x)
  • dNTP Mix (10 mM each): 1 µL (final concentration: 200 µM each)
  • 5mC control primers (10 μM): 2 µL (final concentration: 0.2 µM each)
  • Bisulfite-treated DNA (from preparation step): 1 µL
  • PfuTurbo Cx DNA Polymerase (2.5 U/µL): 1 µL (final concentration: 2.5 U/50 µL PCR)
  • Milli-Q water to 50 µl
  • Final volume: 50 µL

Set up and run the PCR program as follows:
  • Cycle 1: 95 °C for 2 min
  • Cycle 2-41: 95°C for 30 s, 57 °C for 30 s, and 72 °C for 1 min
  • Cycle 42: 72 °C for 10 min

7) Verify PCR amplification by running 5-10 μL of PCR reaction on agarose gel (single band at approx: 300 bp)
8) Use 2 μL of PCR reaction in Zero Blunt TOPO PCR Cloning Kit (Invitrogen) to isolate individual clones for sequencing.
Recommended: Pick and sequence at least 20 individual clones.
9) Check the sequencing results to estimate the conversion efficiency of 5mC at CpG positions.
DNA methylated with CpG methyltransferase selectively at CpG sites is recommended as the spike-in control. Over 90% conversion of 5mC should be obtained.

Restrictions For Research Use only
Handling Advice Tet1 protein is sensitive to temperature, multiple times of freeze and thaw should be avoided.
Storage -80 °C
Expiry Date 3 months
Product cited in: Lister, Mukamel, Nery, Urich, Puddifoot, Johnson, Lucero, Huang, Dwork, Schultz, Yu, Tonti-Filippini, Heyn, Hu, Wu, Rao, Esteller, He, Haghighi, Sejnowski, Behrens, Ecker: "Global epigenomic reconfiguration during mammalian brain development." in: Science (New York, N.Y.), Vol. 341, Issue 6146, pp. 1237905, 2013 (PubMed).

Jiang, Zhang, Wang, Wang, Zhang, Li, Yang, Ma, Sun, Cai, Zhang, Huang, Yu, Wang, Liu, Wu, He, Zhang, Ci, Liu: "Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos." in: Cell, Vol. 153, Issue 4, pp. 773-84, 2013 (PubMed).

Yu, Hon, Szulwach, Song, Zhang, Kim, Li, Dai, Shen, Park, Min, Jin, Ren, He: "Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome." in: Cell, Vol. 149, Issue 6, pp. 1368-80, 2012 (PubMed).