Dinh:MAB Protocol
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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.