Dinh:OxBS Protocol

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OxBS protocol[edit]

  • Cambridge Epigenetix (CEGX) TrueMethyl kit: although each reaction costs > $100, they can have maximum input of only about 1 ug per reaction provided in the kit. We may need to invest in purchasing individual components of the TrueMethyl kit & then making other components ourselves. The most important component in the kit in the oxidant reagent. Since the oxidant reagent is a solution made of KRuO4 and NaOH we can make this solution & always use a control DNA to quantify the efficiency of conversion.
  • Reagents to buy:
 KRuO(4) from Alpha Aesar, $146
 Micro Bio-Spin® Columns with Bio-Gel® P-6 (pack of 25) from BioRad, $96.19
    (Maximum loading per column: 50 ng/uL x 75 uL = 3750 ng)
 EpiTect Bisulfite Kit (48) from Qiagen, $228.34
 5-Hydroxymethylcytosine dNTP Mix from Zymo Research, $70.56
 KAPA Hifi Uracil ReadyMix from KAPA Biosystems, $236.7
 Primers for preparing digestion spike-in controls from idtdna (no special modifications).. $20(?)

Preparation of genomic DNA for BS-seq and oxBS-seq[edit]

  • While the Nature Protocol reference states that DNA preparation for targeted sequencing does not need to be sonicated, the TrueMethyl application note protocol 03 recommends Covaris shearing the genomic DNA to 10 kbp before moving on.

Needle shearing method instead of Covaris shearing[edit]

  • Cheap way to shear the DNA
  • File:Smrtcommunity needle shearing 25kbp.pdf
  • UV the syringe needle
  • Passing a solution of 1-10 ug of genomic DNA through a 26 gauge syringe needle can also shear the DNA.
    • Pass at least 5 times to get 25 kbp fragments. Can run a gel to check.

Shear DNA with QIAEXII beads[edit]

  • Covaris tubes didn't shear well, decided to use QIAEXII beads instead.
  • Follow beads purification and add vigorous vortexing for 90s.

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

  • Use 1.6X AMPure XP beads to purify and concentrate the DN. Wash the beads with 80% ethanol twice and let air dry for 30 minutes after the last wash. Elute the DNA with 22.5 uL of nuclease-free water.
  • Prepare a BioRad Micro Bio-Spin P-6 (preferably SSC buffer) for preoxidation purification.
    • centrifuge tube at 1000 g for 120 s
    • wash 3 times with 500 uL water at 1000 g and 60 s
    • wash 1 last time with 500 uL water at 1000 g and 120 s
    • Add DNA sample to the column, spin at 1000 g for 240 s. Retain the eluate.

Preparation of the oxidant solution[edit]

  • Molecular weight of KRuO(4) is 204.17 g/moles: 0.004897879 moles in 1 g. One 1 g of KRuO(4) is $146.
    • To get 0.015 moles/L, add ~ 3.265 mL of 0.05 NaOH to 10 mg of KRuO4.
  • Add 1.86 uL x 0.2155 M KRuO4 + 1 uL 1 M NaOH + 17.14 uL water = 20 mM KRuO4

Oxidation workflow[edit]

  • Thaw & mix the 1x oxidant reagent by vortexing and centrifuging at 1000 g for 5 s at room temperature. Return the thawed solution to ice.
  • Denature the dsDNA, add to a new 1.5 mL tube the buffer exchanged dsDNA sample, 1 M NaOH and water as detailed below:
 Reagent                               Volume per reaction
 1 ug buffer exchanged dsDNA           Variable
 Nuclease-free water                   up to 21.75
 1 M NaOH                              1.25 uL (total will be 23 uL)
  • Incubate the DNA denaturing solution at 37C in a shaking incubator for 30 minutes.
  • Snap-cool the solution by placing the tube in an ice-water bath (0C) for 5 minutes.
  • While the denatured DNA solution is in the ice-water bath, add 2 uL of fresh 1x oxidant reagent. Briefly vortex and centrifuge for 5s at room temperature. Replace to ice-water bath.
  • Incubate the oxidation reaction at 40C for 30 minutes. Briefly vortex twice, after 10 min, and after 20 min.
  • The solution should remain orange after the incubation. Any other color implies the oxidant reacted with contaminants in the solution. If other colors are observed, re-start the procedure from buffer exchange step.
  • Prepare the PreCR repair mix:
 Reagent                Volume per reaction
 dNTP (10 mM)           0.4 uL
 10X ThermoPol buffer   4.0 uL
 100X NAD+              0.4 uL
 PreCR mix              1.0 uL
 Water                  7.95 uL
  • Add 13.75 uL of PreCR repair mix to each sample and incubate 37C for 30 min.

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.