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]