Samuel J Gurr, PhD

Preparation of MeDIP-enriched, Bisulfite Converted Illumina Libraries

Preparation of MeDIP-enriched, Bisulfite Converted Illumina Libraries

Written 20130513 by Sam White

Adapted from:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763296/

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013100

Reagents:

  • TE

  • phenol:chloroform:IAA, 25:24:1

  • 5x MeDIP Buffer

  • 3M sodium acetate (pH = 5.2)

  • MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS)

  • Proteinase K

  • anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b)
  • Protein A/G Plus Agarose beads (Santa Cruz Biotech)
  • 100% EtOH
  • 70% EtOH

Personal Protective Equipment (PPE):

  • Gloves

Equipment:

Sonication Tube spinner

Procedure

  • Total Time: 3 days
  • Cost/sample:

Notes:

Both MeDIP Buffers should be made up from liquid stocks of each individual component, as each individual component are common molecular biology stock reagents that all lab members should have at their benches.

Do NOT attempt to make the MeDIP Buffers by weighing out and dissolving powdered chemicals for each individual component; it cannot be done accurately with the small quantities required.

This is a multi-day procedure. Read through the protocol thoroughly to plan your time properly.

DNA Isolation

  • Isolate gDNA and resuspend final DNA pellet in TE or H2O. (Initial incubation with Proteinase K is dependent on tissue type and available time to perform procedure.)

  • Quantify gDNA yield and quality. Procedure requires a minimum of 6ug, but more can’t hurt.

DAY 1

  1. Fragment gDNA using sonication
    • Note: fragmentation protocol requires a sample volume of 120µL.
  2. Run 1µl of fragmented on an Agilent Bioanalyzer to verify/quantify size. Successful fragmentation will appear as X

If fragmentation is successful, proceed

  1. Bring fragmented gDNA sample to a volume of 350µL with TE.
  2. Heat sample at 95°C for 10mins and immediately place on ice for 5mins.
  3. Add 100µL of 5x MeDIP Buffer (50mM Na2HPO4, 700mM NaCl, 0.25% Triton-X 100)
  4. Add 45µL of TE
  5. Add 5µL (5µg) of anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b).
  6. Incubate overnight at 4°C rotating end-over-end.

#DAY 2

  1. Wash calculated volume of Protein A/G Plus Agarose beads (Santa Cruz Biotech; need 20µL per sample) with 1x MeDIP Buffer.
  2. Mix stock Protein A/G Plus Agarose beads well and transfer needed volume to clean tube.
  3. Pellet beads by spinning 1000g for 2mins at 4°C
  4. Discard supernatant
  5. Resuspend beads in 1mL of 1x MeDIP Digestion Buffer
  6. Discard supernatant
  7. Resuspend beads in 1mL of 1x MeDIP Digestion Buffer
  8. Discard supernatant
  9. Resuspend in 1x MeDIP Buffer to a final volume of 40µL per sample.
  10. Add 40µL of resuspended Protein A/G Plus Agarose beads to each sample and continue incubation with end-over-end rotation at 4°C for 2hrs.
  11. Pellet the Protein A/G beads by spinning at 1000g for 2mins at 4°C.
  12. Remove and retain supernatant which contains the unmethylated DNA fraction.
  13. Wash beads with 1mL 1x MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS) by repeating steps 9 & 10.
  14. Wash two more times.
  15. Save supernatant which contains the unmethylated DNA fraction each wash.
  16. Resuspend beads in 250uL MeDIP Digestion Buffer.
  17. Add 70ug of Proteinase K and incubate 24hrs @ RT with end-over-end rotation.

Note: The source protocols say to incubate the Proteinase K digest at 55°C. However, if you don’t have a means to do so (since you need a rotator to keep the agarose beads in suspension), according to various sources, Proteinase K retains >80% of it’s enzymatic activity between 20°C - 50°C. So, allow the digest to run longer than recommended (24h).

DAY 3

  1. Add a volume of phenol:chloroform:IAA (25:24:1) equal to your sample volume, vortex throughly and spin at 16,000g for 10mins at room temperature.
  2. Transfer aqueous phase to clean tube. If original sample had cloudy interphase repeat Step X until interphase is no longer cloudy.
  3. Precipitate the DNAs by adding 1/10th volume of 3M NaOAC (sodium acetate; pH = 5.2), 2.5 volumes of 100% EtOH (ethanol), mix throughly and incubate at -20°C for at least 20mins. Note: If expecting low yields, addition of 20ug of glycogen can help improve recovery.
  4. Pellet DNA by spinning samples at 16,000g for 20mins at 4°C.
  5. Discard supernatant and wash pellet with 1000uL 70% EtOH.
  6. Re-pellet DNA by spinning samples at 16,000g for 20mins at 4°C.
  7. Discard supernatant, pulse spin, discard residual supernatant and briefly air dry pellet for 5min at room temperature.
  8. Resuspend methylated DNA in 50µL TE.
  9. Resuspend each unmethylated DNA fraction in 25µL and then pool.
  10. Quantify DNAs