DRIP

DNA:RNA immunoprecipitation

Material

Lysis bufer
100 mM NaCl
10 mM Tris pH 8
25 mM EDTA pH 8
0.5 % SDS
10ug/ml Proteinase K (add fresh)

10x Binding buffer
100 mM Na3PO4 pH 7*
1.4 M NaCl
0.5 % Triton-X

(*) 1M Na3PO4 pH7 (can be prepared by adding 39 ml NaH2PO4 (276 g/L) with 61 ml of Na2HPO4 (284 g/L) and 100 ml H2O)

TE buffer
10 mM Tris pH8
1 mM EDTA

ELution buffer
50 mM Tris pH8
10 mM EDTA
0.5 % SDS

Genomic DNA extraction and digestion

1. Wash the cells (6 well plate/ per condition) with 1X PBS
2. Harvest cells by trypsinization, spin and wash pellet with 1X PBS.
3. Pellet down the cells again by centrifugation (300g/5min/4°C).
4. Resuspend in 500ul lysis buffer and incubate at 37-45°C for at least 3 hrs with rotation (start the experiment in the morning, so that you will have longer incubation time with Proteinase K digestion. The longer incubation the better it is. Do not incubate at more than 45°C), if you have large number of cells forming a clump, pass the clump through a needle 0.7mm. Lysis can be done also overnight (sometime better).
5. Perform Phenol-CHCl3 extraction of nucleic acids as follows:
   • Add 500 μl Phenol-CHCl3 (pH=8.2, use the bottom layer) and gently invert the tube 4-5 times. Centrifuge at high speed (15000rpm/10min/4°C).
   • Cut 1-ml tip and collect the upper phase into another tube
   • Add 500 μl CHCl3 (to get rid of residual phenol) and gently invert the tube 4-5 times. Centrifuge at high speed (15000rpm/10min/4°C).
   • Add 500 μl isopropanol (with 10% of 3M Sodium acetate pH5.2 to increase yield of DNA precipitation) to the collected upper phase and gently invert the tube 4-5 times. Centrifuge (15000rpm/30min/4°C) and collect DNA pellet
   • Wash the DNA pellet with 1ml 75% EtOH and centrifuge (15000rpm/15min/4°C).
   • Discard EtOH (remove with 1ml pipette, briefly spin tubes, and collect the residual EtOH by 200ul pipette) and dry the nucleic acid with an open tube at 37°C (roughly 1 hour, but check regularly)
6. Add DNase/RNase free water until 500 μl and 50μl 10X Fast Digest buffer (Thermo scientific) and let the tube stay at 37°C for about 15min or so
7. Add 20U each of EcoRI, BamHI, HindIII, BsrgI, and XhoI and incubate overnight at 37° C

RNaseH Digestion and purification (version 1)

1. Divide the samples into two halves (~275 μl) and add 27.5 μl RNaseH buffer in each tube and 40U RNaseH in one of the tubes.
2. Compensate for the volume of RNaseH in non-RNH digestible samples by adding DNase/RNase-free water. Incubate all samples at 37°C for 3-4 hours with rotation Add RNase/DNase-free water to all samples to make a final volume of 500 μl and perform phenol-chloroform extraction as step 3
3. Dissolve the pellet in 100 μl RNase/DNase-free water and quantify the yield by nanodrop.

RNaseH Digestion and purification (version 2, preferred)

1. Perform phenol-chloroform extraction as step 3 and quantify yield by nanodrop.
2. Take 5 μg purified product and resuspend in a 135 μl of TE (make duplicate)
3. To one replicate add 15 μl of RNase H buffer and 40U RNaseH (comercial) or 2 μl RNase H (homemade). Fill tubes without RNase up to 150 μl with TE. Incubate all samples at 37°C for 3-4 hours with rotation.

S9.6 DRIP and qPCR

1. Take 5 μg purified product and resuspend in a 500 μl of TE (version 1) or fill the tubes up to 500 μl with TE (version 2). Mix well and take out 50 μl in a separate tube; this will be the input (10%). Add 50 μl 10X binding buffer to the purified products. Different protocols work with various ratios of DNA-antibody beads.
2. Add 3 μg of S9.6 antibody in the remaining 500 μl sample and incubate at 4°C/overnight/rotation (I dilute antibody in 20 μl of 1x binding buffer for easier pipetting)
3. Wash protein A/G magnetic beads in 1x binding buffer. Use magnetic rack to pull down beads and add 1ml of buffer
4. Add 25 μl protein A/G magnetic beads in the IP tubes and incubate at 4°C for 2 hours with rotation
5. Use a magnetic rack to pull down the beads in the washing steps.
6. Wash the beads two times with 1ml 1X-binding buffer (15 min/wash).
7. Wash it once more with TE buffer
8. Add 500 μl elution buffer (+7 μl 20 mg/ml proteinase K), and incubate at 55°C for 45 min
9. Collect supernatant in a fresh tube and perform phenol-chloroform extraction as step 3, add 1uL of glycogen in the step d. Dissolve the pellet by adding 50 μl TE buffer, and keep it on ice for 15-30 min. Pipette gently
10. Perform QPCR with 2 μl of the sample

qPCR (96 WP or 384 WP)

1. Thaw Roche SYBR Green Mastermix (if thawing new, make 1 mL aliquots)
2. Thaw primers (10 μM)
3. Take a well plate and mark some lines that can help you with orientation. For each condition, do a triplicate (important). Think well how to organise it, so it is easy to pipette.
4. Prepare qPCR reaction
   • Add Sybr Green into wells, use repeater pipette
   • prepare mastermix of primers and water (prepare at least 10% more, there is a lot of dead volume) and add it to the wells (try not to touch drop which is already there to avoid cross contamination)
   • add DNA as last (for that I use normal pipette), try not to touch drops already in wells, I pipette it on different sides

qPCR mix for 96 WP (10 μL)
SYBR Green Mastermix 5 μL
Primer fw 0.5 μL
Primer rv 0.5 μL
DNA 2 μL
Water 2 μL

qPCR mix for 384 WP (8 μL)
SYBR Green Mastermix 4 μL
Primer fw 0.4 μL
Primer rv 0.4 μL
DNA 1.2 μL
Water 2 μL

1. Seal well plate with film
2. Spin shortly 1 min at 1000 rpm (centrifuge next to qPCR machine)
3. Perform qPCR (45-50 cycles), use SYBR green template, but adjust annealing temperatures
4. Calculate Ct values from the software (Analysis/Abs quantification/Calculate)
5. Copy Ct values to an excel file and save