ChIP was performed from staged 2-4 hours embryos collected in population cages as described in PMID 17406543 and PMID 17322397 with minor modifications. Briefly, embryos were cross-linked in 1.8% formaldehyde and homogenized in Buffer A1 (15 mM Hepes pH 7.5, 15 mM NaCl, 60 mM, KCl, 4 mM MgCl, 0.5% Triton X-100, 0.5 mM DTT and protease inhibitors). Nuclei were resuspended in A2 buffer (15 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS, 0.5% N-lauroylsarcosine and protease inhibitors) and sonicated to obtain chromatin fragments with an average size of ~500 bp. For input controls, 50 μl of chromatin was used. About 700µg to 1mg of chromatin was incubated with 1.5µg anti-H3K36me3 antibody (abcam, ab9050) overnight at 4ºC. 30 µl of sheep anti-rabbit IgG Dynabeads was added to each chromatin/antibody solution and incubated for 2 hr, 4ºC, then beads were washed 5 times with RIPA Buffer (50 mM Hepes pH 7.5, 0.5 M LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate) and once with 50mM NaCl in TE. Bound complexes were eluted twice with 200 μl of elution buffer (50mM Tris pH 8.0, 10 mM EDTA, 1% SDS) at 65ºC for 30min. The eluate were treated with RNase A (0.2 μg/μl) for 1 hour at 37ºC followed by Proteinase K treatment (0.2 μg/μl) for 1 hour at 55ºC. Crosslinks were reversed by incubating samples at 65ºC overnight.
Growth protocol
dKDM4A mutant with P element insertion (y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}Kdm4AKG04636) was obtained from Bloomington Stock Center at Indiana University (stock #13828). P element KG04636 was mobilized by crossing the mutant to y1w*; CyO, H{w[+mc]=PΔ2-3}Hop2.1/Bc1EgfrE1 flies. Males of KG04646 over the transposase gene PΔ2-3 were crossed to a CyO balanced stock. P element excision was screened by eye color and confirmed by PCR. To generate the dKDM4A genomic rescue, a fragment containing the dKDM4A locus (incl. 1.6 KB upstream / 220 bp downstream) was amplified from genomic DNA of Oregon R flies. Double FLAG tags were added at dKDM4A C-terminus. V423A mutation was generated using Quick Change II XL Site-Directed Mutagenesis Kit (Stratagene); fragment was cloned into pCa4B vector [PMID:18311141]. Site-specific integration at attP40 landing site (2L 25C7) was carried out by Genetic Services. To rescue the mutant, the transgene (FLAG-dKDM4A or dKDM4A-V423A) on the second chromosome was recombined to the chromosome carrying KG04636 insertion.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Label
Cy5
Label protocol
Immunoprecipitated (IP) samples and input samples (200 ng) were amplified by ligation-mediated PCR (LM-PCR ), and 2 µg each were differentially labeled by random-primed, Klenow-based extension as described in PMID 17406303.
ChIP was performed from staged 2-4 hours embryos collected in population cages as described in PMID 17406543 and PMID 17322397 with minor modifications. Briefly, embryos were cross-linked in 1.8% formaldehyde and homogenized in Buffer A1 (15 mM Hepes pH 7.5, 15 mM NaCl, 60 mM, KCl, 4 mM MgCl, 0.5% Triton X-100, 0.5 mM DTT and protease inhibitors). Nuclei were resuspended in A2 buffer (15 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS, 0.5% N-lauroylsarcosine and protease inhibitors) and sonicated to obtain chromatin fragments with an average size of ~500 bp. For input controls, 50 μl of chromatin was used. About 700µg to 1mg of chromatin was incubated with 1.5µg anti-H3K36me3 antibody (abcam, ab9050) overnight at 4ºC. 30 µl of sheep anti-rabbit IgG Dynabeads was added to each chromatin/antibody solution and incubated for 2 hr, 4ºC, then beads were washed 5 times with RIPA Buffer (50 mM Hepes pH 7.5, 0.5 M LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate) and once with 50mM NaCl in TE. Bound complexes were eluted twice with 200 μl of elution buffer (50mM Tris pH 8.0, 10 mM EDTA, 1% SDS) at 65ºC for 30min. The eluate were treated with RNase A (0.2 μg/μl) for 1 hour at 37ºC followed by Proteinase K treatment (0.2 μg/μl) for 1 hour at 55ºC. Crosslinks were reversed by incubating samples at 65ºC overnight.
Growth protocol
dKDM4A mutant with P element insertion (y1 w67c23; P{y+mDint2 wBR.E.BR=SUPor-P}Kdm4AKG04636) was obtained from Bloomington Stock Center at Indiana University (stock #13828). P element KG04636 was mobilized by crossing the mutant to y1w*; CyO, H{w[+mc]=PΔ2-3}Hop2.1/Bc1EgfrE1 flies. Males of KG04646 over the transposase gene PΔ2-3 were crossed to a CyO balanced stock. P element excision was screened by eye color and confirmed by PCR. To generate the dKDM4A genomic rescue, a fragment containing the dKDM4A locus (incl. 1.6 KB upstream / 220 bp downstream) was amplified from genomic DNA of Oregon R flies. Double FLAG tags were added at dKDM4A C-terminus. V423A mutation was generated using Quick Change II XL Site-Directed Mutagenesis Kit (Stratagene); fragment was cloned into pCa4B vector [PMID:18311141]. Site-specific integration at attP40 landing site (2L 25C7) was carried out by Genetic Services. To rescue the mutant, the transgene (FLAG-dKDM4A or dKDM4A-V423A) on the second chromosome was recombined to the chromosome carrying KG04636 insertion.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Label
Cy3
Label protocol
Immunoprecipitated (IP) samples and input samples (200 ng) were amplified by ligation-mediated PCR (LM-PCR ), and 2 µg each were differentially labeled by random-primed, Klenow-based extension as described in PMID 17406303.
Hybridization protocol
A mixture of Cy5-labeled IP DNA and Cy3-labeled input DNA (4.5 µg each for replicates 1, and 9 µg each for replicates 2) was hybridized at 65ºC for 40 hours to Drosophila Whole Genome ChIP-on-Chip Set 244K arrays using Agilent Technologies CGH protocol and reagents (Oligo aCGH Hybridization Kit; Cat. No.: 5188-5220 and Oligo aCGH Wash Buffer Kit; Cat. No.: 5188-5226).
Scan protocol
Slides were scanned with an Agilent High-Resolution DNA Microarray Scanner (G2505C), and image analysis was performed with Agilent Technologies Feature Extraction software (version 10.5.1.1).
