SUPPLEMENTAL MATERIALS AND METHODS
Chromatin immunoprecipitation
Cells were washed with PBS, centrifuged and stored at -80°C. Frozen cross linked cells were
resuspended in lysis buffer (1% SDS and 10 mM EDTA) with protease inhibitor cocktail tablet
(Roche, Basel, Switzerland). Sonication was performed using Sonicator Ultrasonic Processor
S3000 (Misonix, Farmingdale, NY) to generate chromatin fragments smaller than 1 Kb. During
sonication, samples were cooled in an ice water bath. Chromatin fragments were collected by
microcentrifugation at maximum speed for 10 min at 4C. Supernatant was diluted 1:1 in dilution
buffer (70 mM Hepes, pH 7.5, 2.5 mM NaCl, 1.5 mM EDTA, 1.5% Triton and 0.6%
deoxycholate). Diluted chromatin fragments were precleared by adding 20 µl of protein A
agarose beads with salmon sperm DNA (Millipore, Billerica, MA) and incubated at 4C for 1hr.
Supernatant was then immunoprecipitated with anti-H3K4me3 (clone MC315, Millipore,
Billerica, MA), anti-Phospho-NF-κB p65 (Ser536) (Cell Signaling, Danvers, MA), anti-H3K27me3
(Abcam, Cambridge, MA) or normal mouse or rabbit IgG (Santa Cruz, Santa Cruz, CA) at 4C
overnight on a rotation wheel. 20 µl of protein Agarose beads with salmon sperm DNA were
then added into the immunoprecipitation tube and incubated at 4C for 1hr before collecting
beads. Agarose was spun down and washed twice with 1ml of immunoprecipitation (IP) wash
buffer 1 (20 mM Tris-HCl pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS),
once with 1ml of IP wash buffer 2 (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1%
IGEPAL, 1% deoxycholic acid). Immune complexes were eluted twice from the beads by adding
200 μL of IP elution buffer (100 mM NaHCO3, 1%SDS). 20 μL of 5 M NaCl were added to
samples and incubated at 65°C over night. After incubating with RNase A (10 µg/mL) at 37 C
for 30 min and then with protease K (20 mg/mL) at 50 C for 60 min. DNA was recovered using
phenol chloroform extraction and ethanol precipitation at -20°C overnight together with 5 μg of
glycogen. DNA pellets were washed with 70% ethanol air-dried and resuspended in 10-20 μL of
water.
Capture deep sequencing of genomic DNA
Briefly, 1 µg genomic DNA was sheared into fragments of approximately 300-500 bp.
Fragments were End-Repaired and an extra “A” base was added to the 3’ end. Illumina index
adapters were ligated to the fragments and 10 cycles of PCR amplification were applied to each
sample after ligation. DNA was quantified by PicoGreen assay (Invitrogen, Carlsbad, CA).