Dinh/Dinh 2015/NOTES/2015-9-28

From ZhangLabWiki
Jump to navigation Jump to search

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