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For suspension cells, ensure the appropriate number are resuspended in 10 ml of fresh media to which 37% of formaldehyde solution is subsequently added until a final concentration of 1% is reached. Vortex for a few seconds to displace from the bottom and incubate for 10 minutes at room temperature in agitation.
Grind frozen tissue into powder with a pestle and mortar. Pour the resulting powder into a 15 or 50 ml falcon. Add 10 ml PBS + 270 μl formaldehyde 37% (final concentration 1%) to the frozen powder. Vortex for a few seconds to displace from the bottom, next incubate for 10 minutes at room temperature in agitation.
Block the reaction with 500 μl Glycine 2.5 M (final concentration 0.125 M). Incubate for 5 minutes at room temperature.
Transfer the cells to a 50 ml falcon and centrifuge at 2500 rpm for 5 minutes at 4ºC.
Discard the supernatant and wash twice with ice-cold PBS ph 7.4 and centrifuge at 2500 rpm for 5 minutes at 4ºC after each washing.
Resuspend cells in 5 ml Cell Lysis Buffer supplemented with protease inhibitor. To facilitate the cell membrane breaking, pass the lysate 3 times to a douncer. Incubate for 15 minutes at 4ºC.
Centrifuge at 4000 rpm for 5 minutes at 4ºC. Discard the supernatant.
Resuspend nuclei in Cell Lysis Buffer. Pipette with cut tips to homogenize better.
Divide the sample into small aliquots and sonicate for 15 minutes (high power; 30 seconds sonication, 30 seconds rest). Put ice into the sonicator to avoid sample overheat.
Centrifuge at 12000 rpm for 10 minutes at room temperature to remove nuclear debris. Discard the pellet. Repeat this passage until the pellet cannot be detected. Store the samples at -20ºC. If SDS precipitates, dissolve it prior to centrifuge. Take an aliquot of chromatin to quantify (2–3 μl) and to assess the size (30–40 μl).
De-crosslink chromatin by incubating samples at 65ºC for 4 hours (results may improve with an overnight incubation).
Incubate for 30 minutes with Proteinase K 50 μg/ml final concentration at 42ºC.
Add 1 volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1). Mix with vortex and let samples stand at room temperature for 2–3 minutes.
Centrifuge at 12000 rpm for 5 minutes and transfer the aqueous phase to a new tube. (If the interphase is dirty repeat steps 3 and 4).
Add 1/5 volume of AcNH4 10 M and 2.5 volumes of EtOH 100%. Mix and let the DNA precipitate for at least 30 minutes at -20ºC.
Centrifuge at max speed for 15 minutes at 4ºC. Discard the supernatant.
Wash with 70% ethanol and centrifuge at max speed for 15 minutes at 4ºC. (Try to discard as much supernatant as you can without touching the pellet.)
Resuspend with TE buffer and pipette until complete dissolution.
Incubate for 30 minutes at 37ºC with RNAse A at a final concentration of 50 μg/μl.
Prepare a 1.5% agarose gel.
|0.1% SDS (protein interaction dependent)|
|1.1% Triton X-100|
|16.7mM Tris HCl, pH 8.1|
Take 70 μl of magnetic beads for each sample to be immunoprecipitated. (Take the extra volume in excess: 0.5–1 times more.)
After precipitation with a magnet, discard the supernatant and wash twice with 600 μl at 5% BSA/PBS.
After the second wash reconstitute the initial volume (70*Nº of samples μl). (Take the extra volume in excess: 0.5–1 times more.)
Take 20 μg (dependent on the tissue/cell type) for each sample, dilute the chromatin 1:10, and bring to a final volume of 1 ml with dilution buffer.
Take 25 μl of beads for each sample and add them to the chromatin for the pre-clearing step.
Divide the remaining beads into 45 μl aliquots. Add the corresponding antibody to each tube, plus a negative control (specific IgG). Incubate overnight at 4ºC in a rotating wheel.
|Table 8||Table 9|
|Low Salt Buffer||High Salt Buffer|
|Tris HCl 50mM, pH 8.0||Tris HCl 50mM, pH 8.0|
|150mM NaCl||500mM NaCl|
|0.1% SDS||0.1% SDS|
|1% NP40||1% NP40|
|1mM EDTA||1mM EDTA|
|0.5% Deoxycholate Na||0.5% Deoxycholate Na|
|Table 10||Table 11|
|LiCl Buffer||TE Buffer|
|Tris HCl 50mM, pH 8.0||Tris HCl 10mM, pH 8.0|
|250mM LiCl||0.25mM EDTA|
|0.1% SDS||Table 12|
|1% NP40||Elution Buffer|
|1mM EDTA||100mM HaHCO3|
|0.5% Deoxycholate Na||1% SDS|
Please Note: From this stage it is better to work with siliconized tubes.
Discard the beads from the chromatin samples by putting the tubes in the magnet.
Wash the Ab-Bead complexes twice with ice-cold 300 μl at 5% PBS/BSA. Spin after the second wash and remove the supernatant.
Add 1 ml of the chromatin to each sample and resuspend with the tip. Incubate for 2 hours at 4ºC in a rotating wheel. Store the exceeded chromatin for the input sample.
Spin the samples and put them on the magnet.
Wash twice with 1 ml low salt buffer.
Wash twice with 1 ml high salt buffer.
Wash twice with 1 ml LiCl buffer.
Wash twice with 1 ml TE when adding the second washes. Change tubes for new ones.
Remove last wash almost completely with the pipette.
Prepare Elution Buffer (EB) and set the thermomixer to 65ºC.
Add 100 μl of Elution Buffer to each sample. Incubate for 10 minutes at 65ºC in the thermomixer.
Put the supernatant in a new tube and repeat step 11 to obtain a final volume of 200 μl.
Take 50 μl of the exceeded chromatin from the pre-clearing as a 5% input. Add 150 μl Elution Buffer to reach a 200 μl final volume.
Incubate samples and inputs at 65ºC overnight to de-crosslink.