Dinh:TAB Protocol
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TAB-Seq protocol[edit]
- 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 QIAEX II gel extraction kit (150), $172 - These columns are cheaper than QiaQuick and the MinElute columns, but seems to have better flexibility in purifying larger fragments - Since I do not want to perform Covaris shearing on the DNA for BSPP, I should use the QIAEX II columns instead of the QiaQuick/MinElute columns.
IMPORTANT note for Tet1[edit]
- 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.
Preparation of genomic DNA for TAB-seq[edit]
- For each 1 ug of genomic DNA, add 10 ng of control DNA
Glucosylation[edit]
- Protect 5hmC with beta-GT as follows:
Reagent Volume for one reaction 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 AmPure XP beads.
Oxidation: NgTet1 (NEB)[edit]
- Dilute Fe solution 1/250 with nuclease free water to a working 2 mM solution
- Make 125 uL: 0.5 uL x 500 mM FeSO4 + 124.5 uL nuclease free water
- Prepare the NgTet1 oxidation reaction:
T4-BGT treated DNA (500 ng) 15.5 uL 10x NgTet1 buffer 2.0 uL 2 mM Fe Solution 0.8 uL 48.3 uM NgTet1 (lot6) 1.7 uL
- Mix well and allow th reaction to proceed at 34C for 30 minutes.
- Add 1 uL proteinase K (20 mg/mL) to each reaction
- 37C for 30 minutes then 95C for 10 minutes.
- Increase volume to 75 uL and purify with Micro-Bio-spin 30 columns
- Purify with AmPure Beads. Elute in 30 uL EB.
Oxidation: mTet1 (WiseGene)[edit]
- Prepare the mTet1 oxidation reaction:
T4-BGT treated DNA (500ng) 13.25 uL Tet oxidation reagent 2 7.50 uL Tet oxidation reagent 1 1.75 uL Tet1 protein 2.50 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.
- Purify with AmPureXP beads. Elute in 30 uL EB buffer
- The oxidized DNA can be stored at -20C for several weeks.
Oxidation: mTet2 (NEB)[edit]
- Dilute Fe solution 1/250 with nuclease free water to a working 2 mM solution
- Make 125 uL: 0.5 uL x 500 mM FeSO4 + 124.5 uL nuclease free water
- Prepare the mTet2 oxidation reaction:
T4-BGT treated DNA (500 ng) 13.5 uL 5x mTet2 buffer 5.0 uL 2 mM Fe Solution 2.5 uL 63.6 uM mTet2 (lot16) 4.0 uL
- Mix well and allow the reaction to proceed at 37C for 60 minutes.
- Add 1 uL proteinase K (20 mg/mL) to each reaction
- 37C for 30 minutes then 95C for 10 minutes.
- Increase volume to 75 uL and purify with Micro-Bio-spin 30 columns
- Purify with AmPureXP Beads. Elute in 30 uL EB.
Bisulfite conversion[edit]
- Perform bisulfite conversion with the EpiTect bisulfite kit.
- Use the low input protocol (15 uL of DNA protect buffer)