genotype: RelA-/- cell type: Mouse Embryonic Fibroblast treatment: TNF-α for 30 mins
Treatment protocol
30 minutes TNF-alpha
Extracted molecule
genomic DNA
Extraction protocol
MEF’s were treated with TNF-α (20 ng/ml) for the indicated times. DNA was cross-linked for 10 minutes with 1% formaldehyde and stopped in 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. Purified chromatin was sonicated to ~500 bp using the Bioruptor (Diagenode, Inc). The lysate was then centrifuged to remove cell debris, diluted 1:2 in IP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.0), precleared for 15 minutes with 10 ml StaphA beads, and incubated overnight with 5 mg of either anti-Sirt6 (Abcam, ab62739), RelA (Abcam, ab7971) or Rabbit IgG. After incubation with 10 ml StaphA beads for 15 minutes at 4C, the immune complexes were collected by centrifugation and washed 2x with Dialysis Buffer (2 mM EDTA, 50 mM Tris-Cl pH 8.0, 0.2% Sarkosyl) and 3x with IP wash buffer (100 mM Tris-Cl pH 9.0, 500 mM LiCl, 1% NP40, 0.1% Deoxycholic Acid). The protein-DNA complexes were eluted from the beads in 100 µl elution buffer (1% SDS, 50 mM NaHCO3) for 15 minutes followed reverse cross-linking by addition of 5M NaCl and incubating overnight at 65C, followed by 100mg/mL RNAseA digestion. DNA was purified with the Ziagen Mini-elute Reaction Cleanup Kit and amplified using the Whole Genome Amplification kit (Sigma) as described by the manufacturer.
Label
Cy5
Label protocol
Each DNA sample (1 μg) was denatured in the presence of Cy5- or Cy3-labeled randon nonamers and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix (6 mM each in TE buffer) for 2 hours at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8), precipitated with isopropanol, and resuspended in water.
Channel 2
Source name
Input DNA from RelA-/- Mouse Embryonic Fibroblast cells treated with TNF-alpha for 30 minutes
genotype: RelA-/- cell type: Mouse Embryonic Fibroblast treatment: TNF-α for 30 mins
Treatment protocol
30 minutes TNF-alpha
Extracted molecule
genomic DNA
Extraction protocol
MEF’s were treated with TNF-α (20 ng/ml) for the indicated times. DNA was cross-linked for 10 minutes with 1% formaldehyde and stopped in 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. Purified chromatin was sonicated to ~500 bp using the Bioruptor (Diagenode, Inc). The lysate was then centrifuged to remove cell debris, diluted 1:2 in IP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.0), precleared for 15 minutes with 10 ml StaphA beads, and incubated overnight with 5 mg of either anti-Sirt6 (Abcam, ab62739), RelA (Abcam, ab7971) or Rabbit IgG. After incubation with 10 ml StaphA beads for 15 minutes at 4C, the immune complexes were collected by centrifugation and washed 2x with Dialysis Buffer (2 mM EDTA, 50 mM Tris-Cl pH 8.0, 0.2% Sarkosyl) and 3x with IP wash buffer (100 mM Tris-Cl pH 9.0, 500 mM LiCl, 1% NP40, 0.1% Deoxycholic Acid). The protein-DNA complexes were eluted from the beads in 100 µl elution buffer (1% SDS, 50 mM NaHCO3) for 15 minutes followed reverse cross-linking by addition of 5M NaCl and incubating overnight at 65C, followed by 100mg/mL RNAseA digestion. DNA was purified with the Ziagen Mini-elute Reaction Cleanup Kit and amplified using the Whole Genome Amplification kit (Sigma) as described by the manufacturer.
Label
Cy3
Label protocol
Each DNA sample (1 μg) was denatured in the presence of Cy5- or Cy3-labeled randon nonamers and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix (6 mM each in TE buffer) for 2 hours at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8), precipitated with isopropanol, and resuspended in water.
Hybridization protocol
12 μg of Cy5-labeled ChIP sample and 6 μg of Cy-3 labeled total sample were mixed, dried and resuspended in 40 μL of buffer including hybridization buffer (Nimblegen), alignment oligonucleotides (Nimblegen) and Component A (Nimblegen). The labeled DNA samples were next denatured and hybridized to “MM8” arrays overnight at 42°C. Samples were co-hybridized with input DNA as a reference.
Fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NIMBLESCAN 3.0 extraction software (Nimblegen). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. To normalize across samples, the biweight mean of this log2 ratio was subtracted from each point. ChIP-chip time courses were done twice or more for most time points; representative data from single time courses are shown.
