DNA-RNA immunoprecipitation (DRIP) protocol

A step-by-step DRIP protocol, including R-loop preparation and associated reagents.

DNA-RNA immunoprecipitation (DRIP) uses the S9.6 anti-RNA-DNA hybrid antibody to capture RNA-DNA hybrids along chromosomes. DRIP is typically followed by mapping  DNA fragments on a few loci or even across the whole genome with qPCR, microarray hybridization, or deep sequencing.

Thanks to Professor Frédéric Chédin’s lab at UC Davis for providing us with this protocol.


R-Loop preparation


Reagents

25 mM rNTP stock (NEB N0466S) - dilute to 2.5 mM rNTP for experiment

T3 RNA Polymerase, 50 U/µL (NEB M0378S)

10 x RNAPol Reaction Buffer (NEB M0378S)

1M DTT (from frozen stock, made in house)

2.5% Tween-20 (diluted in water, made in house)

pCALM3_2 plasmid ( pCALM3_2 carries an R-loop forming portion of the human CALM3 gene)

RNase A, 10 mg/mL (DNase free) – dilute to 1.0 mg/mL for experiment

RNase H, 5 U/µL (NEB M0297S)

Proteinase K, 10 mg/mL (Sigma P2308)

DuRed (nucleic acid dye), 1000X (Brigen D009)

ApaLI, 2500 units (NEB R0507S)


Protocol

  1. Mix:
    pCALM3_2 2 µg
    10x buffer 10 µL
    1M DTT 2 µL
    2.5% Tween-20 2 µL
    2.5 mM rNTP 10µL
    H20 to 99.4 µL total
  2. Initiate reaction by adding 0.6 µL of T3 RNA Polymerase, mix gently, and split into two reactions (50 µL each). Incubate at 37°C for 10 minutes.
  3. Inactivate enzyme by adding 1 µL of 10 mM EDTA.
  4. To one sample, add 10 µL of 0.1 mg/mL RNase A, and the other (negative control) add 10 µL 0.1 mg/mL RNase A and 4 µL RNase H. Incubate for 30 minutes at 37°C.
  5. Add 4 µL of Proteinase K and incubate for 30 minutes at 37°C.
  6. Cleaned up on Axygen PCR purification kit and eluted in 50 µL ddH2O, separately.
  7. Each sample (2 samples totally) split into two tubes, put one tube on ice, the other one was used for the following digestion.
  8. Digest:
    25 ul DNA
    5 µL 10X Cutsmart buffer NEB
    1 µL Ll ApaLI NEB
    19 µL ddH2O
    50 µL total
  9. 37°C 2 h.
  10. Cleaned up on Axygen PCR purification kit and eluted in 25 µL ddH2O. Four samples (2–5) plus one pCALM3_2 plasmid (1) were obtained for DRIP experiment.
  11. In order to verify that R-loop formation did occur, load ~200 ng on a 0.9% 1x TBE gel WITHOUT nucleic acid dye and run at 90V for 60 min. Use Glycerol at 10% final as a loading dye. Post-stain with DuRed (nucleic acid dye). R-loop formation causes a characteristic shift in mobility compared to un-transcribed or RNase H-treated samples. Results are shown below (Figure 1).

Figure 1. Transcription from pCALM3_2 to generate R-loops. Each digestion reaction was run on an agarose gel. pCALM3_2 carries a portion of the human CALM3 gene that forms R-loops when transcribed with the T3 RNA polymerase. Treatment with RNase A (digests single-stranded RNA) does not affect the R-loops structure (lane 2) whereas treatment with RNase H (digests RNA in DNA-RNA hybrids) destroy R-loops structure (lane 3). pCALM3_2 plasmid can be digested by ApaL restriction enzyme without affecting the R-loop structures.


DNA-RNA hybrid Immunoprecipitation by using antibodies pre-immobilized on beads


Reagents

PBS (phosphate buffer)

Triton X100

ssDNA (Salmon sperm single strand DNA)

Recombinant Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)

Isotype antibody (Mouse (G3A1) mAb IgG1 Isotype Control #5415, 2.5 mg/ml, Mouse IgG1, kappa)

EDTA (Ethylene Diamine Tetraacetic Acid)

2.5% Tween-20 (diluted in water, made in house)

ApaLI (NEB R0507S)

RNase A, 10 mg/mL (DNase free) – dilute to 1.0 mg/mL for experiment

RNase H, 5 U/µL (NEB M0297S)

Proteinase K, 10 mg/mL (Sigma P2308)

DuRed (nucleic acid dye), 1000X (Brigen D009)

Qiagen PCR purification kit


Protocol

Antibodies pre-immobilized on beads

  1. Prepare eight tubes of Protein A beads.
  2. 100 µL protein A beads each tube washed twice in 1 mL of 1X PBS, 0.1% Triton X100, centrifuge 1 min at 3,600 rpm 4°C, carefully aspirate the supernatant.
  3. Each tube resuspended with 1 mL 1X PBS, 0.1% Triton X100 and 7.5 µg ssDNA (Salmon sperm single strand DNA/20 µL beads), shake gently 10 mins at room temperature. Then centrifuge 1 min at 3,600 rpm at 4°C, aspirate the supernatant. Wash once in 1 mL of 1X PBS, 0.1% Triton X100, centrifuge 1 min at 3,600 rpm 4°C, carefully aspirate the supernatant.
  4. 5 µL S9.6 antibody (1mg/mL) (test antibody 5 µg, add to protein A) added to four tubes as positive control and 5 µL isotype antibody (5 µg) added to rest of four tubes as a negative control, make up the samples to 1 mL with 1X PBS, 0.1% Triton X100. Shake gently 10 mins at room temperature.
  5. Wash twice with 1 mL 1X PBS, 0.1% Triton X100, centrifuge 1min at 3,600 rpm 4°C, aspirate the supernatant.
  6. Resuspend each tube in 100 µL PBS, 0.1% Triton X100 and add 1 µL 0.5M EDTA.


Add DNA

  1. Remove 5 µL of each input R-loop (RNase A treated), R-loop (RNase A+H treated), ApaLI digested R-loop (RNase A treated), ApaLI digested R-loop (RNase A+H treated) for gel analysis as control.
  2. Add 35 µL of R-loop (RNase A treated), R-loop (RNase A+H treated), ApaLI digested R-loop (RNase A treated), ApaLI digested R-loop (RNase A+H treated) to two tubes (S9.6, isotype), respectively.
  3. Rotate gently for 2 hours at 4°C.
  4. Centrifuge 1 min at 3,600 rpm 4°C, remove all depleted supernatants and retain for electrophoresis.
  5. Wash three times in 1X PBS, 0.1% Triton X100, centrifuge 1 min at 3,600 rpm 4°C and aspirate the supernatant.
  6. Add 50 µL elution buffer + 5 µL proteinase K, then shake in 1,400 rpm 30 mins 50°C, centrifuge 1 min at 13,000 rpm at room temperature. Collect supernatant.
  7. Clean up by Qiagen PCR purification kit and eluted in 30 µL.

Figure 2. DNA-RNA hybrid Immunoprecipitation (DRIP) data. pCALM3_2 was used to generate R-loops. S9.6 (ab234957) immunoprecipitates R-loops in the presence or absence of prior digestion by ApaLI, which does not affect R-loop structure. Prior treatment with RNase A (digests single-stranded RNA) does not affect the IP signal whereas prior treatment with RNase H (digests RNA in DNA-RNA hybrids) eliminates the signal.


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