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.

Stage 1 - R-loop preparation

Materials and reagents

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

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

10X 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 the experiment

RNase H, 5 U/µL (NEB M0297S)

Proteinase K, 10 mg/mL

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

ApaLI, 2500 units (NEB R0507S)

Steps

Mix:

pCALM3_2 2 µg

10X buffer 10 µL

1M DTT 2 µL

2.5% Tween-20 2 µL

2.5 mM rNTP 10 µL

H₂O to 99.4 µL total

Initiate the 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 min.

Inactivate enzyme by adding 1 µL of 10 mM EDTA.

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 min at 37°C.

Add 4 µL of Proteinase K and incubate for 30 min at 37°C.

Clean up using an Axygen PCR purification kit and elute in 50 µL ddH₂O, separately.

For each sample (2 samples in total), split into two tubes. Put one tube on ice, and use the other for the following digestion.

Digest:

25 µl DNA
5 µL 10X Cutsmart buffer NEB
1 µL Ll ApaLI NEB
19 µL ddH₂O
50 µL total

Incubate at 37°C for 2 h.

Clean up using the Axygen PCR purification kit and elute in 25 µL ddH₂O.

You should have four samples (2–5) plus one pCALM3_2 plasmid (1) for the DRIP experiment.

To confirm that the R-loop formation has occurred, load ~200 ng of your sample on a 0.9% 1X TBE gel without nucleic acid dye and run at 90 V for 60 min.

Use 10% Glycerol 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 (Figure 1).

Stage 2 - DNA-RNA hybrid immunoprecipitation using antibodies pre-immobilized on beads

Materials and reagents

PBS

Triton X100 or NP-40

Salmon sperm single strand DNA (ssDNA)

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

Mouse IgG1, kappa monoclonal [MOPC-21] - isotype control (ab18443)

Ethylene Diamine Tetraacetic Acid ( EDTA )

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 the experiment

RNase H, 5 U/µL (NEB M0297S)

Proteinase K, 10 mg/mL

DuRed (nucleic acid dye), 1,000X (Brigen D009)

Qiagen PCR purification kit

Steps

Prepare eight tubes of Protein A beads. Add 100 µL of protein A beads to each tube and wash twice in 1 mL of 1X PBS and 0.1% Triton X-100 (0.1% NP-40 can be used instead) by centrifuging for 1 min at the manufacturer's recommended speed at 4°C. Carefully aspirate the supernatant each time.

Resuspend the beads in each tube with 1 mL 1X PBS, 0.1% Triton X-100 (0.1% NP-40 can be used instead), and 7.5 µg ssDNA (20 µL beads), and shake gently for 10 min at room temperature.

Centrifuge for 1 min at the manufacturer's recommended speed at 4°C, then aspirate the supernatant. Wash once in 1 mL of 1X PBS, 0.1% Triton X-100 (0.1% NP-40 can be used instead), and centrifuge 1 min at the manufacturer's recommended speed at 4°C, carefully aspirate the supernatant.

Add 5 µL S9.6 antibody (1 mg/mL, test antibody 5 µg), to the beads in one tube and 5 µL isotype antibody (5 µg) to the remaining four tubes as a negative control. Make the samples up to 1 mL using 1X PBS with 0.1% Triton X-100 (0.1% NP-40 can be used instead).

Shake gently for 10 min at room temperature.

Wash twice with 1 mL 1X PBS with 0.1% Triton X-100 (0.1% NP-40 can be used instead), centrifuge for 1 min at the manufacturer's recommended speed at 4°C, and aspirate the supernatant.

Resuspend each tube in 100 µL PBS with 0.1% Triton X-100 (0.1% NP-40 can be used instead) and add 1 µL 0.5M EDTA.