DAF-seq Protocol

This protocol covers the complete DAF-seq workflow from nuclei isolation through DNA extraction. For primer design guidelines, see Primer Design. For SsDddA and DddI purification, see Protein Purification. After library preparation and sequencing, use the DAF-QC Pipeline for data processing and quality control.

Reagents & Buffers

Buffer A

Final ConcentrationStock ConcentrationVolume stock soln
RNase- DNase- free H2O100%959 uL
15 mM Tris-Cl, pH 8.01 M Tris-Cl, pH 8.015 uL
15 mM NaCl5 M NaCl3 uL
60 mM KCl3 M KCl20 uL
1 mM EDTA, pH 8.00.5 M EDTA, pH 8.02 uL
0.5 mM EGTA, pH 8.00.5 M EGTA, pH 8.01 uL
0.5 mM Spermidine0.5 M Spermidine1 uL
10 nM ZnCl210 uM ZnCl21 uL

Note: Buffer A without spermidine or ZnCl2 can be prepared in large quantities (e.g. 100 mL) and safely stored for up to 6 months at room temperature. Add 1 uL of 0.5 M spermidine and 1 uL of 10 uM ZnCl2 to 998 uL of buffer A stock right before using. Store spermidine at -20 C for up to 6 months.

Additional reagents

  • Reagents for nuclei isolation, e.g.
    • Buffer A + 0.1% (w/v) digitonin
    • 1x PBS, pH 7.4 + 0.5% BSA
  • UNG inhibitor (NEB M0281)
  • SsDddA stock: 100 uM, aliquots stored at -80 C
  • DddI stock: 1000 uM, aliquots stored at -80 C
  • NEB Monarch Spin gDNA Extraction Kit (T3010)
  • Qubit dsDNA assay
  • LoBind tubes

Nuclei Isolation

Nuclei isolation is cell and tissue-type dependent and should be optimized by the user to ensure completion. Below is an example protocol which we have used for LCLs. Triton and IGEPAL can be substituted for digitonin.

  1. Collect 500k-1M cells into a 1.5 mL LoBind tube.
  2. Pellet at 350 x g for 5 min at 4 C. Remove supernatant.
  3. Wash cells with 1 mL 1x PBS, pH 7.4 + 0.5% BSA.
  4. Pellet at 350 x g for 5 min at 4 C. Remove supernatant.
  5. Resuspend pellet in 60 uL Buffer A.
  6. Add 60 uL 2x Lysis Buffer. Mix by gently tapping the side of the tube and incubate 10 min on ice.
  7. Spin 350 x g for 5 min at 4 C. Remove supernatant.
  8. Resuspend nuclei in 50 uL Buffer A.

IMPORTANT: Ensure that cells are fully lysed before proceeding to the next step.

  1. Count 100k-250k nuclei and add Buffer A + 1 uL UNG inhibitor (2 U) to a final volume of 47 uL.

On-Nuclei Cytosine Deamination

  1. Add 2 uL SsDddA (100 uM stock, final 4 uM) to the 47 uL nuclei suspension.
  2. Incubate 10 min at 25 C. Mix gently; avoid vortexing.
  3. Immediately add 1 uL DddI (1000 uM, 5-molar excess) to stop the reaction.
  4. (Optional) Store at -20 C before DNA extraction.

DNA Extraction

Follow the manufacturer's instructions. The steps below reflect the lab's working sequence for samples after the on-nuclei deamination stop. For routine targeted DAF-seq applications, we recommend NEB Monarch Spin gDNA Extraction Kit (T3010) as the length of isolated DNA is typically sufficient for the amplification of 2-7 kb products. A HMW kit may be necessary for longer range PCR products.

  1. Add PBS to bring the total volume to 100 uL (typically 50 uL).
  2. Add 1 uL Proteinase K and 3 uL RNase A; mix by brief vortexing.
  3. Add 100 uL Cell Lysis Buffer; vortex 10 s.
  4. Incubate 5 min at 56 C in a thermal mixer with agitation (~1400 rpm).
  5. Add 400 uL gDNA Binding Buffer; vortex 5-10 s.
  6. Centrifuge briefly (~1 min at 16,000 x g) if needed to collect contents; transfer to a gDNA purification column seated in a collection tube.
  7. Close cap and centrifuge 3 min at 1,000 x g to load/bind DNA onto the column.
  8. Immediately centrifuge 1 min at 16,000 x g to clear the membrane.
  9. Discard collection tube and place column into a fresh collection tube.
  10. Add 500 uL gDNA Wash Buffer; centrifuge 1 min at >=12,000 x g.
  11. Discard flow-through and repeat the 500 uL Wash Buffer step and spin.
  12. Transfer column to a clean 1.5 mL LoBind tube.
  13. Add 35 uL pre-warmed (56 C) gDNA Elution Buffer directly to the membrane.
  14. Let stand 1 min at room temperature; centrifuge 1 min at >=12,000 x g to elute DNA.
  15. Store eluted gDNA at 4 C (short-term) or -20 C (long-term). Quantify with Qubit.

The DNA is now ready for PCR.

Notes

  • The reaction volume of the "On-Nuclei Cytosine Deamination" may be doubled to accommodate 500K cells. If 100 uL volume is used, no additional buffer is required at step 14 of "DNA Extraction".
  • We have observed that the recovery of SsDddA-treated DNA is approximately 10-20% as efficient as untreated DNA using the Monarch Spin gDNA Extraction Kit. This recovery is typically sufficient for downstream applications.

Next steps

After library preparation and sequencing, process your data with the DAF-QC Pipeline to assess targeting efficiency, deamination rates, strand calling, and other quality metrics.