1. First “fast” dissection:
Dissect for 20 minutes third instar larvae in ice-cold PBS and remove gut and fat body but leave carcass attached \(allows easier handling). Dependingon dissection speed this results in 100 to 200 carcasses per dissection round. For wild type samples we used the fly strains: Canton S or w118.
After one round of dissection fix carcasses with attached discs immediately for 20 min at room temperature by gently mixing in 1 ml cross-linkingsolution \(1.8 % formaldehyde, 50 mM Hepes pH 8, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl). The cross-linking solution is changed 3-4 times during fixation.Stop the cross-linking by washing for 3 min in 1ml PBS/0.01 % Triton X-100/125 mM glycine with 3-4 washes.
Optionally: During the 20-minute fixation start another round of dissections.
3. Stop fixation and wash:
Wash fixed carcasses for 10 min in 1 ml wash A \(10 mM Hepes pH 7.6, 10 mM EDTA, 0.5 mM EGTA, 0.25 % Triton X-100) and subsequently for 10 min in 1 mlwash B \(10 mM Hepes pH7.6, 200 mM NaCl, 1m M EDTA, 0.5 mM EGTA, 0.01 % Triton X-100) changing the wash solution 3-4 times. Store carcasses at 4 ºCin wash B \(maximal over night).
4. Second “fine” dissection of fixed carcasses:
Dissect brain and disc tissues off the carcasses in wash B and pool approximately 400 to 800 brain-disc complexes for one chromatin sample.
5. Lysis and sonication:
Perform lysis and sonication of dissected material \(400 brain disc complexes) in 0.5 ml RIPA buffer \(140 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 %Triton X-100, 0.1 % SDS, 0.1 % sodium deoxycholate, 1 mM PMSF, 0.5 % N-Laurylsarcosine \(only added for the sonication), 1 x Complete protease inhibitorcocktail \(Roche)) in two steps. First sonication is conducted 2 times for 2 min with a tip sonicator \(Omni-Ruptor 250 \(Omni-International Inc.),microtip, power output: 20, pulser: 60).
The material is then transferred to a glass round bottom Covaris sonication tube \(TC12x24, 520056) and sonicated 3 times for 10 min in a Covarismachine \(Covaris S, maximum all). \(You can include 5 small porcelain beads to increase sonication efficiency \(Precellys Keramik Kügelchen, 1.4 mm,Peqlab, 91-PCS-CK14P.)
This sonication yields genomic DNA fragments of 500 bp for 80 % of the chromatin. \(We cannot bring down the residual unfragmented DNA by furtherincreasing the sonication times.)
6. Spin and freeze chromatin:
Remove insoluble material by centrifugation at 4 ºC for 20 min at 16000 g. Dilute and aliquot the soluble chromatin in the supernatant.Quick-freeze the diluted aliquots in liquid nitrogen and store at -80 ºC. Avoid repeated freeze-thaw cycles.
Dilution: dilute chromatin with RIPA buffer \(without N-Laurylsarcosine) to 1 brain disc complex per 18 ul \(e.g. top up 400 brains in 0.5 ml up to 7.2ml with RIPA).
1. Sample set up:
Use 35-100 μl of diluted chromatin to quantify by qPCR after reversal of cross-links \(treated from RNase step onwards as described below), to obtain anestimate of the chromatin concentration \(input DNA). For immunoprecipitations, 110 or 250 μl of sonicated chromatin solution is taken up in 1 ml RIPAbuffer \(140 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 % Triton X-100, 0.1 % SDS, 0.1% sodium deoxycholate, 1 mM PMSF, Roche 1 x complete).
2. Pre-clearing extracts:
Prepare 50 % bead suspension by equilibrating 0.1 g Protein A Sepharose CL-4B beads \(GE-Healthcare, cat no. 17078001) in 1 ml RIPA \(with all proteaseinhibitors), incubate at 4 ºC for 1 h, spin at 4 ºC for 10 min at 16000 g and re-suspend in 500 ul RIPA. Pre-clear chromatin by adding 50 %v/v beads suspension and incubate at 4 ºC for 1 h \(rotating).
3. Antibody incubation:
After centrifugation at 4 ºC for 30 sec at 16000 g transfer the chromatin to fresh tubes \(1.5 ml Eppendorf) and incubate with specific antibodies\(anti-L\(3)mbt1211-1477aa and anti-dSfmbt531-980aa) at 4 ºC over night \(12-16 h).
Generally we use 5 to 10 ug of antibody per ChIP reaction. However, the amount needs to be optimized experimentally for each antibody.
IgGs of the species the antibody was raised in \(e.g. rabbit IgG, abcam, ab37415);
ideally: chromatin prepared from knocked down or mutant for the protein of interest. \(We used the modENCODE criteria to validate the L\(3)mbt antibodyin western blot and immunofluorescence, for modENCODE data standards see link to this document:
Positive controls to test ChIP procedure:
Antibodies to histone modifications work well in ChIPs \(e.g. from abcam).
4. Bead incubation:
Recover antibody-chromatin complexes by incubation with 40 ml of 50 % v/v beads suspension at 4 ºC for 3 h.
Wash at 4 ºC, 10 min with 1 ml as follows:
5 x RIPA
1 x LiCl \(250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 % NP-40, 0.5 % sodium deoxycholate)
2 x TE \(10 mM Tris-HCl pH 8.0, 1 mM EDTA)
Remove each time the solution by centrifugation at 4 ºC for 30 sec at 16000 g.
6. RNase/proteinase K digest and cross-link reversal:
After the last TE wash remove supernatant and resuspend beads in 100 μl TE. Incubate with 50 μg/ml RNase \(Roche, cat no. 11119915001 orFermentas, EN0531) at 37 ºC for 30 min. Quickly spin samples down.
Then adjust to 0.5 % SDS, 10 mM CaCl2, 0.5 mg/ml proteinase K \(Fermentas, EO0491) and incubate at 55 ºC for 1 hr. For reversal ofcross-links, samples were incubated at 65 ºC \(350 rpm if using shaking heat block) for 6 h or over night. \(Cover the heat block \(i.e. withalufoil) to improve isolation of the tubes, as this will minimize condensation, which could alter the concentrations in the sample.)
7. Phenol/Chloroform extraction:
Perform phenol/chloroform extraction using Phase-Lock heavy gel tubes \(Eppendorf, cat no. 32005152) following manufacturer’s instructions.
Briefly: Transfer sample to 2 ml Phase-Lock tube and add 1 x sample volume \(approximately 120 ul if you followed the protocol above) ofPhenol-Chloroform \(Phenol stabilized : Chloroform : Isoamyl alcohol 25 : 24 : 1, AppliChem, A0837), shake 5 min at room temperature \(don’t vortexthe Phase-Lock tubes\!), spin 5-10 min at 13,200 rpm in phase-lock tubes. Transfer upper phase to fresh 1.5 ml eppendorf tube.
8. EtOH precipitation:
To precipitate DNA, add 1/10 volume of 3 M NaOAc pH 5.2, 2.5 volumes of 100 % cold ethanol and 1 ul of 20 mg/ml glycogen as carrier. Freeze for atleast 30 min at
-80 ºC. Centrifuge at 13,200 rpm for 20 min at 4 ºC and discard supernatant. Wash the pellet with 1 ml of 70 % ethanol, centrifuge at 13,200rpm for 5 min at room temperature and discard supernatant. Air-dry the pellet and resuspend in i.e. 500 ul of DEPC nuclease-free H2O \(AppliedBiosystems, AM9920). Store in small aliquots, i.e. 100 ul at -20 ºC. Avoid freeze-thaw cycles.
Use 10 μl of eluate to check enrichment of specific primers by qPCR reaction.
1. ChIP eluate is analyzed using a real-time PCR instrument \(Applied Biosystems 7500 or CFX96 Real Time System, Bio-Rad) using SYBR Green \(AppliedBiosystems, cat no. 4309155 or Bio-Rad cat no. 170-8880) and standard settings \(Applied Biosystems, Bio-Rad).
2. PCR is performed in duplicates and serial dilutions of purified input DNA are measured together with the immunoprecipitated DNA samples to calculatethe amount of target sequence in immunoprecipitated chromatin relative to the amount of target sequence in input chromatin.
3. With each antibody immunoprecipitation reactions were performed in triplicates and for each PCR fragment, the amount of DNA in theimmunoprecipitated material was expressed as percentage DNA present in the input material.
4. Primer sequences for control regions and Ubx PRE can be found also in Papp and Muller, 2006, Genes Dev, 20, 2041-54.
a) Always use Complete protease inhibitor cocktail and PMSF from lysis onwards \(except for the last two TE washes).
b) Rotate tubes during 4 ºC incubation steps.
c) Can use DNA-low bind Eppendorf tubes to minimize loss of DNA during storage.
d) Theoretically 14 brain disc complexes should suffice for one ChIP reaction. However, if chromatin is prepared from very little tissue ChIPs yieldlower amounts of DNA. Therefore, we always prepare larger batches of chromatin with high DNA concentration during lysis and sonication that are thendiluted for the ChIP reactions.