Dinh/Dinh 2015/NOTES/2015-9-28
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TAB-seq preparation of H1 ESCs (repeat with new T4-BGT)[edit]
T4 BGT glucosylation (Thermo Scientific)[edit]
- Set up (100 uL total volume):
unsheared gDNA 17.94 uL (2.1 ug) 10X UDP-glucose 10.00 uL 10X Epi buffer 10.00 uL Nuclease free H2O 57.77 uL T4 BGT 3.00 uL 5hmC/5mC/C controls 1.29 uL
- Mix gently and spin down for a few seconds.
- Incubate at 37C for 15 minutes.
- Stop the reaction by heating at 65C for 20 min.
- Purify with 1.8X AMPure beads.
- Elute in 40 uL water total. DO NOT elute in EDTA, DNA should be free of EDTA.
- Qubit: 27.9 ng/uL x 40 uL = 1116 ng (53% yield)
- Leave DNA on beads!
Tet treatment[edit]
- Test 2 different Tet analogs.
- Reaction for NgTet1 (NEB), 20 uL, in order:
10X NgTet1 buffer 2.00 uL 48.3 uM NgTet1 1.70 uL T4 BGT treated DNA 15.50 uL 2 mM Fe solution 0.80 uL
- Incubate at 34C for 30 minutes.
- Add 1 uL Proteinase K (20 mg/uL)
- 50C x 60 minutes.
- Clean up with 2X AMPure beads.
- Reaction for mTet2 (NEB), 20 uL, in order:
T4 BGT treated DNA 12.30 uL 5X mTet2 buffer 4.00 uL 63.6 uM mTet2 3.20 uL 2 mM Fe solution 0.50 uL
- Incubate at 37C for 71 minutes.
- Add 1 uL Proteinase K (20mg/uL)
- 50C x 48 minutes
- Clean up with 2X AMPure beads.
- Reaction for mTet1 (Wisegene), 25 uL, in order:
T4 BGT treated DNA 11.00 uL Tet oxidation R2 7.50 uL Tet oxidation R1 1.75 uL Tet1 protein 2.50 uL
- Incubate at 37C for 71 minutes.
- Add 1 uL Proteinase K (20mg/uL)
- 50C x 48 minutes
- Clean up with 2X AMPure beads.
Bisulfite convert with EpiTect Plus kit[edit]
- Elute in 30 uL
- Note: I prepared 2 tubes of oxBS samples, and 1 BS sample for comparison. For the oxBS samples, I purified the oxidized DNA with a BioRad polyacrylamide P6 column prior to bisulfite treatment.
- Qubit:
NgTet 3.02 ng/uL x 30 = 90.6 ng mTet1 2.94 ng/uL x 30 = 88.2 ng mTet2 5.86 ng/uL x 30 = 175.8 ng oxBS#1 8.01 ng/uL x 30 = 240 ng oxBS#2 8.86 ng/uL x 30 = 266 ng BS 13.1 ng/uL x 30 = 393 ng
- The yield was very low this time, I probably need to be more carefully in the AMPure Beads purification (don't overdry, let the beads sit in water for 10 minutes at 55C?, etc.)
Verification of controls[edit]
File:DD 9-28-15 TAB and oxBS controls gel1.jpg File:DD 9-28-15 TAB and oxBS controls gel2.jpg