Dinh/Dinh 2015/NOTES/2015-9-19
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TAB-seq preparation of H1 ESCs[edit]
T4 BGT glucosylation[edit]
- Set up (150 uL total volume):
unsheared gDNA 17.94 uL (2.1 ug) 50X UDP-glucose 3.00 uL 10X NEB Buffer 4 15.00 uL Nuclease free H2O 109.77 uL T4 BGT 3.00 uL 5hmC/5mC/C controls 1.29 uL
- Incubate at 37C for 29 hours.
- 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:
T4 BGT treated DNA 15.50 uL 10X NgTet1 buffer 2.00 uL 2 mM Fe solution 0.80 uL 48.3 uM NgTet1 1.70 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), 25 uL:
T4 BGT treated DNA 13.50 uL 5X mTet2 buffer 5.00 uL 2 mM Fe solution 2.50 uL 63.6 uM mTet2 4.00 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:
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.
- Qubit:
NgTet1 treated 9.21 ng/uL x 40 uL = 378 ng (87.5% yield) mTet1 treated 5.80 ng/uL x 40 uL = 238 ng (84%) mTet2 treated 7.38 ng/uL x 40 uL = 303 ng (80%)
Epitect Bisulfite Conversion[edit]
- Set up (final volume is 140 uL):
Treated DNA 40 uL DNA protect buffer 15 uL Bisulfite mix 85 uL
- Incubate O/N using oxBS program.
- Purify according to manual.
- Qubit:
NgTet1 treated 7.70 ng/uL x 30 uL = 231 ng (61%) mTet1 treated 4.16 ng/uL x 30 uL = 125 ng (52.5%) mTet2 treated 7.19 ng/uL x 30 uL = 216 ng (71.3%)
Control DNA preparation[edit]
- See [[1]]