Dinh:MAB Protocol

From ZhangLabWiki
Jump to navigation Jump to search

Protocol for MAB-seq[edit]

Preparation of genomic DNA for MAB-seq[edit]

  • Ideally genomic DNA should be sheared before processing. I will use QIAEX II beads to shear the DNA to ~10kbp.

Add digestion control to the DNA to achieve 1% wt/wt.[edit]

M.sss I treatment[edit]

  • 1 ug of gDNA:
 Reagent       Volume for 1X
 gDNA          30.0 uL
 M. sssI        1.5 uL
 SAM (32mM)     1.0 uL
 10X Mg-free    5.0 uL
 Water         12.5 uL
  • Incubate reaction for 2 hours.
  • Add 0.5 uL M.sssI and 1 uL SAM and incubate at 37C for 4 additional hours.
  • Purify the DNA with QIAEX II beads and repeat the M. sssI treatment for 3 more rounds.
 Each round uses 2 uL of M. sssI, so total of M. sssI use will be 8 uL x 4 U/uL = 32 U for each 1 ug of genomic DNA.
  • Each round of treatment will take 6 hours x 4 = 24 hours minimum.

Bisulfite treatment[edit]

  • Perform bisulfite treatment using oxidized and non-oxidized DNA using the Qiagen EpiTect bisulfite kit according to the protocol "Sodium Bisulfite Conversion of Unmethylated Cytosines in DNA Isolated from FFPE Tissue Samples"
  • Some slight modifications are as followed:
  • Reaction set up:
 Reagent                               Volume per reaction
 DNA solution                           40 uL
 Bisulfite mix (dissolved)              80 uL
 DNA protect buffer                     20 uL
  • Program on thermocycler:
 95 C,  5 min
 60 C, 25 min
 95 C,  5 min
 60 C, 85 min
 95 C,  5 min
 60 C, 175 min
 95 C,  5 min
 60 C, 25 min
 95 C,  5 min
 60 C, 85 min
 95 C,  5 min
 60 C, 175 min
 20 C, HOLD
  • Purify using the manufacturer's instructions.