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DNA-RNA immunoprecipitation (DRIP) protocol

A step-by-step DRIP protocol, including R-loop preparation and associated reagents.
Last edited Fri 19 May 2023

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

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)

Steps

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).

Transcription from pCALM3_2 to generate R-loops

Stage 2 - 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)

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

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

Steps

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.