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= Protocol for MAB-seq = ===Preparation of genomic DNA for MAB-seq=== * 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.=== === M.sss I treatment === * 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=== * 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.
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