5hmC TAB-Seq Kit

Details for Product No. ABIN1741992, Supplier: Log in to see
Application
DNA Sequencing (Seq), Tet-assisted Bisulfite Sequencing (TAB-Seq)
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Purpose The 5hmC TAB-Seq Kit can be used to prepare genomic DNA to differentiate 5-hydroxylmethylcytosine (5hmC) from 5-methylcytosine (5mC) for subsequent sequencing applications by converting 5mC to 5-carboxylcytosine (5caC) while keeping 5hmC protected.
Characteristics Each kit contains enough reagents for up to 3 genome-wide or 9 loci-specific sequencing reactions based on recommended protocols.
Components
  • 10×βGT protection buffer: 30 μL
  • 10 mM UDP-Glucose: 15 μL
  • T4-βGT (40 μM): 7 μL
  • Tet1 oxidation reagent 1: 50 μL
  • Tet1 oxidation reagent 2: 180 μL
  • Tet1 protein: 45 μL
  • Proteinase K (20 mg/mL): 15 μL
Material not included
  • 5mC spike-in control DNA and primers
  • 5hmC spike-in 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 2.5 ng (0.25 %) 5hmC control DNA, then shear the mixed genomic DNA to average 400 bp.

Assay Procedure

βGT-based 5hmC protection
1) Prepare the β-glucosyltransferase (βGT)-based reaction.

  • Genomic DNA from preparation step: 2-5 µ, up to 17.1 µL (final concentration: 100-250 ng/µL)
  • 10 mM UDP-Glucose: 0.4 µL (final concentration: 200 µM each)
  • 10×βGT protection buffer: 2 µL (final concentration: 1x)
  • T4-βGT (40 μM): 0.5 µL (final concentration: 1 µM)
  • Milli-Q water to 20 µL
  • Final volume: 50 µL

Typically, 2-3 μg genomic DNA to start with in Step 3 should be sufficient.
2) Mix well and allow the reaction to proceed at 37 °C for 1 h.
3) Purify DNA using QIAquick PCR Purification Kit (Qiagen). Elute DNA in 50 μL water.
70-80 % DNA from last step can be recovered after next step.

mTet1-based 5mC oxidation
4) 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: 4.5 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 (last step in this section).
Note: If a different amount of genomic DNA is applied, change the volume of the reaction accordingly (from 20 μL to 50 μ;) 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.
5) Mix well and allow the reaction to proceed at 37 °C for 1 h.
6) Add 1 μL Proteinase K (20 mg/mL) to the reaction mixture and incubate at 50 °C for 1 h.
7) 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
8) Apply 50 ng Tet1-oxidized DNA from Step 7 to EpiTect Bisulfite Kit (Qiagen) or MethylCode Bisulfite Conversion Kit (Invitrogen) following the manufacture’s instruction.
9) 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 step 8): 2-4 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

5mC control primer sequences:
Forward: 5’-TTTGGGTTATGTAAGTTGATTTTATG
Reverse: 5’-CACCCTACTTACTAAAATTTACACC

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

10) Verify PCR amplification by running 5-10 μL of PCR reaction on agarose gel (single band at approx. 300 bp)
11) 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.
12) 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 95 % conversion of 5mC should be obtained.

Test 5hmC protection efficiency of 5hmC spike-in control DNA
13) 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)
  • 5hmC control primers (10 μM): 2 µL (final concentration: 0.2 µM each)
  • Bisulfite-treated DNA (from step 8): 2-4 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

5hmC control primer sequences:
Forward: 5’-GTAGATTGTATTGAGAGTGT
Reverse: 5’-TACCCAACTTAATCGCCTTG
Note: For the reverse primer design, assume that ‘C’s on control are 100 % of 5hmC and will not be deaminated after bisulfite treatment.
14) Set up and run the PCR program as follows:
  • Cycle 1: 95 °C for 2 min
  • Cycle 2-41: 95°C for 30 s, 51 °C for 30 s, and 72 °C for 1 min
  • Cycle 42: 72 °C for 7 min

15) Verify PCR amplification by running 10 μL of PCR reaction on agarose gel (single band at approx. 200 bp)
16) 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.
17) Check the sequencing results to estimate the protection efficiency of 5hmC on control.
Over 80 % of 5hmC should be read as C. The real protection efficiency should be over 95 % due to contamination of most commercially available 5hmdCTP. If experiments are run alongside conventional bisulfite treatments, dCTP contamination can be adjusted for by direct measurement of bisulfite converted cytosine in the 5hmC spike-in control.

The Genomic DNA prepared from section: mTet1-based 5mC oxidation is now ready for genome-wide or loci-specific 5hmC sequencing analysis following the following recommended protocol.

Recommended Protocol For Genome-Wide 5hmC Sequencing Analysis
1) 500 ng-900 ng treated genomic DNA (from section: mTet1-based 5mC oxidation) was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina TruSeq Genomic DNA adapters) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adapter to insert.
2) Adapter ligated fragments with 200-600 bp inserts were gel purified by 2 % agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen).
3) Bisulfite treated adapter-ligated DNA was amplified by PCR with PfuTurbo Cx Hotstart DNA polymerase. The number of PCR cycles used was determined by quantification of bisulfite treated adapter-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20 nM.
4) Final sequencing libraries were purified with AMPure XP beads or 2 % agarose gel electrophoresis and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries).
Up to 3 separate PCR reactions were performed per sample.

Recommended Protocol For Loci-Specific 5hmC Sequencing Analysis<1) Use the table below as a guide to prepare 50 μL PCR:
  • 10×HotStarTaq PCR buffer : 5 µL (final concentration: 1x)
  • dNTP Mix (10 mM each): 1 µL (final concentration: 200 µM each)
  • primers of interested loci (12.5 μM each): 1 µL (final concentration: 0.25 µM each)
  • Bisulfite-treated DNA (from step 8): 2 µL
  • HotStarTaq DNA Polymerase (5 U/ μL): 0.25 µL (final concentration: 1.25 U/50 µL PCR)
  • Milli-Q water to 50 µl
  • Final volume: 50 µL

Protocol is provided for HotStarTaq DNA Polymerase. Other polymerase or PCR protocols can be substituted. However, Hot Start polymerase is recommended.
2) Set up and run the PCR program as follows:
  • Cycle 1: 95 °C for 16 min
  • Cycle 2-36: 95°C for 30 s, 50-68 °C for 30 s, and 72 °C for 1 min
  • Cycle 42: 72 °C for 7 min

3) Verify PCR amplification by running 5-10 μL of PCR reaction on agarose gel (clear single band)
4) Purify the PCR product with QIAquick PCR Purification Kit (Qiagen). Elute DNA in 30 μL water or EB.
5) Apply the purified PCR products to Sanger sequencing analysis. The presence of ‘C’ represent the positions of 5-hmC.

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
Supplier Images
 image for 5hmC TAB-Seq Kit (ABIN1741992) 5hmC TAB-Seq Kit
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).