Cells were crosslinked on tissue culture plates following replacement of growth medium with 1X PBS (25 mls for 15 cm plate). For suspension cells, cells were centrifuged (500g) and resuspended in 1X PBS. Formaldehyde was added to a final concentration of 1% and incubated for 10 min at room temperature with mixing. Crosslinking was quenched by adding glycine to a final concentration of 0.125 M. Crosslinked cells were centrifuged (900g), resuspended in 1 ml 1X PBS, and transferred to 1.6 ml eppendorf tube and centrifuged. Cells were then resuspended in 1 ml cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40) and incubated on ice for 15 minutes. Cells were centrifuged (900g) and resuspended in 1 ml nuclear lysis buffer (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS). 0.6 ml of IP dilution buffer (20 mM Tris pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS) was added and the chromatin sample was transferred to a 14 ml polypropylene round-bottom tube (Falcon). Sonication was performed on ice with a Misonix 3000: 3 minutes total time, 1 second ON, 1 second OFF, Output level setting 3.0. Sonicated chromatin was transferred to 1.6 ml eppendorf tube centrifuged (16,000g) and supernatant was transferred to 3.4 mls IP dilution buffer. Chromatin samples were pre-cleared for 2 hours with 50 ul Protein G beads (50:50 mixture) with 50 microgram control IgG. During this time, IP antibodies (5 micrograms) were pre-bound to 35 ul Protein G beads in 1 ml 1X PBS for 2 hours. Pre-bound antibodies were centrifuged (5000g) and supernatant discarded. Pre-cleared chromatin was centrifuged (5000g) and supernatant transferred to a new tube. 180 ul of input material (3.6%) was set aside. 1.25 ml of chromatin was then transferred to pre-bound antibodies (corresponding to chromatin from 5x106 cells) and incubated 2 hours or overnight. Beads were then washed once with 1 ml IP wash 1 (20 mM Tris pH 8.1, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt wash buffer (20 mM Tris pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash 2 (10 mM Tris pH 8.1, 1 mM EDTA, 0.25M LiCl, 1% NP-40, 1% deoxycholic acid), and twice with TE. Chromatin was then eluted twice with 100 ul elution buffer (100 mM sodium bicarbonate, 1% SDS). 2 ug of RNase A and 12 ul of 5 M NaCl were added to the 200 ul elution and also to input sample, mixed, and incubated overnight at 65 degrees to reverse crosslinks. The next day, 60 micrograms of Proteinase K were added and incubated at 42 degrees for 2 hours. Samples were then diluted with TE to a final volume of 400 microliters. Samples were phenol:chloroform:IAA extracted (twice for input) then chloroform extracted. 10 micrograms of glycogen were then added followed ethanol precipitation. Pellets were then washed with 70% ethanol, dried on 70 degree heat block, then resuspended in deionized water (60 microliters for IP, 120 microliters for input).
Label
Cy5
Label protocol
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Cells were crosslinked on tissue culture plates following replacement of growth medium with 1X PBS (25 mls for 15 cm plate). For suspension cells, cells were centrifuged (500g) and resuspended in 1X PBS. Formaldehyde was added to a final concentration of 1% and incubated for 10 min at room temperature with mixing. Crosslinking was quenched by adding glycine to a final concentration of 0.125 M. Crosslinked cells were centrifuged (900g), resuspended in 1 ml 1X PBS, and transferred to 1.6 ml eppendorf tube and centrifuged. Cells were then resuspended in 1 ml cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40) and incubated on ice for 15 minutes. Cells were centrifuged (900g) and resuspended in 1 ml nuclear lysis buffer (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS). 0.6 ml of IP dilution buffer (20 mM Tris pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.01% SDS) was added and the chromatin sample was transferred to a 14 ml polypropylene round-bottom tube (Falcon). Sonication was performed on ice with a Misonix 3000: 3 minutes total time, 1 second ON, 1 second OFF, Output level setting 3.0. Sonicated chromatin was transferred to 1.6 ml eppendorf tube centrifuged (16,000g) and supernatant was transferred to 3.4 mls IP dilution buffer. Chromatin samples were pre-cleared for 2 hours with 50 ul Protein G beads (50:50 mixture) with 50 microgram control IgG. During this time, IP antibodies (5 micrograms) were pre-bound to 35 ul Protein G beads in 1 ml 1X PBS for 2 hours. Pre-bound antibodies were centrifuged (5000g) and supernatant discarded. Pre-cleared chromatin was centrifuged (5000g) and supernatant transferred to a new tube. 180 ul of input material (3.6%) was set aside. 1.25 ml of chromatin was then transferred to pre-bound antibodies (corresponding to chromatin from 5x106 cells) and incubated 2 hours or overnight. Beads were then washed once with 1 ml IP wash 1 (20 mM Tris pH 8.1, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt wash buffer (20 mM Tris pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash 2 (10 mM Tris pH 8.1, 1 mM EDTA, 0.25M LiCl, 1% NP-40, 1% deoxycholic acid), and twice with TE. Chromatin was then eluted twice with 100 ul elution buffer (100 mM sodium bicarbonate, 1% SDS). 2 ug of RNase A and 12 ul of 5 M NaCl were added to the 200 ul elution and also to input sample, mixed, and incubated overnight at 65 degrees to reverse crosslinks. The next day, 60 micrograms of Proteinase K were added and incubated at 42 degrees for 2 hours. Samples were then diluted with TE to a final volume of 400 microliters. Samples were phenol:chloroform:IAA extracted (twice for input) then chloroform extracted. 10 micrograms of glycogen were then added followed ethanol precipitation. Pellets were then washed with 70% ethanol, dried on 70 degree heat block, then resuspended in deionized water (60 microliters for IP, 120 microliters for input).
Label
Cy3
Label protocol
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Hybridization protocol
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
