Dinh:TAB Protocol
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TAB-Seq protocol
- This protocol cannot start from methylated adaptor ligated libraries. The 5mC would be converted to T.
- Protocol guides:
Nature protocol for Tet-assisted bisulfite sequencing of 5hmC : http://www.nature.com/nprot/journal/v7/n12/abs/nprot.2012.137.html Manual for kits
- T4 Phage β-glucosyltransferase (T4-BGT) from NEB ($65.63)
T4 Phage β-glucosyltransferase NEB buffer 4 (10x) Uridine Diphosphate Glucose (50X)
- Tet1 Oxidation kit from Wisegene ($669 + $90 shipping)
Tet1 oxidation reagent 1 Tet1 oxidation reagent 2 Tet1 protein Proteinase K
- Misc:
Micro Bio-Spin 30 columns (BioRad) - By gravitational force, larger fragments will get pulled down first, smaller fragments are retained in the gel. - Use P6 columns may be better because it have a smaller pore size, so cleaner separation can be achieved. EpiTect Bisulfite Kit PfuTurbo Cx Hotstart DNA polymerase (agilent) CpG Methyltranferase (M.SssI, NEB), $65
IMPORTANT note for Tet1
- Upon first use, aliquot Tet1 into single use portions. Thaw/Freeze up to only 3 times!
- Same for Tet1 oxidation reagent 1 and 2.
- The reaction solution should turn from transparent to slightly brown after mixing Tet oxidation reagents 1 and 2.
Creating methylated lambda DNA for spike in control
- Set up an in vitro methylation reaction:
Reagent Volume Milli-Q water 44 uL NEBuffer 2 (10x) 10 uL S-adenosylmethionine (SAM) 2 uL Unmethyl- lambda DNA 450ng/ul 40 uL SssI methylase ( 20 U/uL) 4 uL ------------------------------------- Final volume 100 uL
- Mix well and incubate at 37C for 2 hours. Heat inactivate at 65C for 20 min.
- NOTE: Use fresh SAM.
- Clean up the DNA with the QIAEX II gel extraction kit and elute methylated DNA in 20 uL Milli-Q water. (NOTE: Do not use QiaQuick columns because they can only elute up to 10 kbp).
Preparation of genomic DNA
- For each 1 ug of genomic DNA, add 5 ng of methylated lambda DNA control.
- Fragment the DNA (recommended to use Covaris):
- For RRBS may use MspI digestion to fragment the DNA
- For BSPP may use needle shearing to get 25 kbp fragments
- May add 5hmC control generated by PCR for the oxBS protocol to sheared DNA.
- Purify sheared DNA with the Qiagen MinElute columns
Preparation of Tet-assisted oxidation
- Protect 5hmC with beta-GT as follows:
Reagent Volume Sheared DNA, 1-3 ug variable UDP-glucose, 50X 1 uL NEBuffer 4, 10X 5 uL Nuclease-free water to 49 uL T4-BGT, 10U/uL 1 uL ------------------------------------- Total volume 50 uL
- Mix well and incubate at 37C for 12 to 18 hours.
- Clean up using 1.8X AMPure beads.
- Prepare the mTet1 oxidation reaction:
Reagent Volume 500 ng glucosylated DNA variable, up to 27 uL Nuclease-free water to 27 uL Tet oxidation reagent 2 15 uL Tet oxidation reagent 1 3.5 uL Tet1 protein 5.0 uL --------------------------------------- Total 50 uL
- NOTE: 500 ng DNA can yield 200-300 ng after purification
- Mix well and allow the reaction to proceed at 37C for 80 minutes.
- Add 1 uL proteinase K (20 mg/mL) to the reaction mixture and incubate at 50C for 1 hour.
- Clean up using BioRad Micro Bio-Spin P30 columns and then a Qiagen QIAquick column. Elute in 30 uL EB buffer
- The oxidized DNA can be stored at -20C for several weeks.
Bisulfite conversion
- Perform bisulfite conversion with the EpiTect bisulfite kit.