Sequencing of sodium-bisulfite modified genomic DNA originally introduced by M. Frommer (Frommer et al., 1992) is a widely used “gold standard” method for DNA-methylation analysis. Since this method relies on a harsh chemical treatment of DNA it causes a lot of DNA damage and hence a dramatic loss of high quality DNA for PCR amplification and further analysis. In the meantime several commercial kits are available for this procedure which work reasonably well when starting with large amounts of DNA.

Here we describe a protocol for small numbers of cells and little DNA which requires some specific handling. The protocol is based on a strategy originally introduced by our lab (Hajkova et al., 2002) using agarose embedded DNA. This physical trapping helps to avoid DNA loss during the various incubation steps while maintaining a good bisulphite conversion rate. We will introduce two alternative procedures to perform bisulphite treatment of agarose embedded small DNA aliquots or cells and guide through some generally critical points in the bisulphite reaction and primer design.

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We also include tips for the process of data processing after sequencing which is facilitated by a new and very useful software tool (BiQ Analyzer). This tool is freely available and allows rapid and reproducible processing and evaluation of bisulphite sequencing data. It generates standardized table output formats allowing direct database integration.

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Two alternative protocols (A and B) for agarose embedded DNA of cell preparation

In the following we will describe two protocols for bisulphite treatment of small aliquots of mammalian cells/DNA. Both procedures only differ in the first step, i.e. the preparation of the DNA for bisulphite treatment. We routinely use protocol A for collected mammalian cells in the range >100 and B for very scarce material (such as zygotes/early mouse embryos), i.e. few to 100 cells.

Protocol A: Preparation of genomic DNA from samples with more than 100 cells per sample

  1. Mix the collected mammalian cells (>100) with 50 µl of solution A (25 mM EDTA, 75 mM NaCl), 50 µl of solution B (10 mM EDTA, 10 mM Tri-HCl, pH 8.0, 1% SDS), add 2 µl of Proteinase K (20 mg/ml) and 5 µg of yeast t-RNA (use 1.5 ml Eppendorf tubes)
  2. Incubate 1h at 37°C
  3. Add 50 µl of phenol and 50 µl of chloroform/isoamylalcohol (24:1)
  4. Mix the samples gently by inverting the tubes
  5. Centrifuge the samples at 13,000 rpm for 10-15 min
  6. Transfer the supernatant into fresh tubes and add 100 µl of chloroform/isoamylalcohol (24:1)
  7. Mix the samples gently by inverting the tubes
  8. Centrifuge 5 min at 13,000 rpm
  9. Transfer the supernatant into fresh tubes and add another 5 µg of t-RNA and 1/10 volume of 3M sodium acetate (pH5.5), then add 350 µl of 100% ethanol
  10. Precipitate the DNA at -20°C over night or longer
  11. Centrifuge the samples for 30 min at 13,000 rpm at 4°C
  12. Remove the ethanol and let the pellets dry at room temperature
  13. Dissolve the pellet in 4 µl ddH2O over night (do not dissolve the pellets in larger volumes since the DNA is later embedded into one single agarose bead – highest yield; dissolving the pellet over night is necessary to bring the DNA entirely into solution)
  14. Add 0.4 µl of freshly prepared 2 M NaOH and 10 µl of molten 2% LMP agarose (comment 1)
  15. Overlay with 200-500 µl of heavy mineral oil (white, Aldrich)
  16. Boil for 10 min in a water bath
  17. Cool down on ice until the bead is solid
  18. Proceed with bisulphite treatment

Protocol B: Bisulfite treatment in agarose embedded cells

This Protocol should be used as an alternative if you want to avoid precipitation steps, e.g. when working with very low amounts of cells/DNA; few to 100 cells or equivalent DNA content (up to 300 pg).

  1. Add 15 µl of 2% LMP agarose to the sample and overlay with 100-300 µl of heavy mineral oil (note: total volume of the Cell suspension should not exceed 5µl)
  2. Boil the samples for 10 min in a water bath to destroy the cells
  3. Cool the samples for at least 10 min on ice until the bead is solid
  4. Remove the oil (be careful in case if the bead is still not solid)
  5. Add 400 µl of solution A (25mM EDTA, 75 mM NaCl) and 50 µl of Proteinase K (20mg/ml). Centrifuge briefly to make sure that there is no residual oil between the bead and the solution
  6. Incubate at 50°C overnight
  7. Carefully remove the solution as complete as possible
  8. Wash the samples 2x with 400 µl 1xTE for 15 min.
  9. Rinse the bead with 400 µl of 0.3M NaOH
  10. Wash the sample 2x with 400 µl of 0.3M NaOH
  11. Wash the samples 2x with 1xTE
  12. Remove the TE entirely and store the bead at 4°C (no longer than a couple of days)
  13. Proceed with bisulphite treatment

Agarose embedded bisulfite modification

Agarose beads formed according to protocols A or B are used for the following procedure:

  1. Prepare bisulfite solutions freshly as follows: mix 1.9 g sodium metabisulfite (Merck no. 1.06528.0500) with 2.5 ml ddH2O and add 750 µl of freshly prepared 2 M NaOH, dissolve the powder by strong vortexing (it takes time, if the powder does not dissolve it can be done at 50°C). Dissolve 55 mg of hydroquinon (Sigma) in 500 µl ddH2O at 50°C. Mix both solutions when chemicals are dissolved completely. (comment 2)
  2. Add 400 µl of freshly prepared bisulfite solution to each bead (as prepared according to the protocol A or B. Overlay with mineral oil (following protocol B). (When adding the solution to beads from protocol A make sure the beads are in contact with the solution and there is no residual oil between the bead and the solution; brief spin down helps)
  3. Transfer the samples to 50°C and incubate for 3.5 h
  4. Cool the samples briefly on ice (makes the bead better visible and “harder” for solution exchange)
  5. Remove the bisulfite solution and rinse the bead with 400 µl of 1x TE at RT (put 400 µl of 1x TE onto the bead and remove the TE immediately to avoid starting diffusion)
  6. Wash the samples 2x with 400 µl 1xTE for 15 min at RT
  7. Rinse the bead with 400 µl of 0.3M NaOH at RT
  8. Wash the sample 2x with 400 µl of 0.3M NaOH at RT
  9. Wash the samples 2x with 1xTE at RT
  10. Remove the TE entirely and store the bead at 4°C (can be stored for 2 weeks max.)
  11. Proceed with Bisulphite PCR amplification (Bis-PCR)

Bisulphite PCR amplification, cloning and sequencing

For Bisulphite PCR-amplification we regularly use the HotFirePol from Solis BioDyne, Tartu, Estonia. It combines the characteristics of a Hot Start Taq with a good performance und a high cost-efficiency. We experienced that some amplicons may work better with HotStarTaq from Qiagen (has to be tested).

Our standard Bis-PCR protocol runs as follows:

Melt the agarose bead at 70-80°C and transfer 3 µl of the molten agarose into the PCR tube containing the following ingredients (kept at RT or on ice):

  • 5 µl 10xbuffer (Solis BioDyne)
  • 5 µl MgCL2 (25 mM)
  • 4 µl dNTPs (2.5 mM each)
  • 1 µl forward primer (10 µM)
  • 1 µl reverse primer (10 µM)
  • 0.6 µl HotFirePol (5 U/µl)
  • 30.4 µl ddH2O

Transfer into a preheated (95°C) PCR block and start the following program: