Cells were cross-linked for 10 min in media containing 1% formaldehyde before quenching with 1.25mM glycine. Cell pellets were collected and resuspended in cell lysis buffer (1% Igepal, 85mM KCL, 5mM PIPES pH 8) to a concentration of 1mL per 10^7 cells, incubated on ice for 15 mins, and dounced 20x. Nuclear pellets were collected at 430xg for 5min at 4C and resuspended in nuclear lysis buffer (50mM Tris pH8, 10mM EDTA, 1% SDS, protease inhibitors), 20uL per 10^6 cells, and incubated on ice for 30 min. Lysates were sonicated to fragment lengths of 200 to 500bp. The sonicated lysate was spun at 16,100g for 10 mins at 4°C and aliquoted. The chromatin was diluted 5x in IP dilution buffer (50mM Tris pH 7.4, 1mM EDTA, 150mM NaCl, 1% Igepal, 0.25% Na Deoxychlorate and protease inhibitors). 5ug of chromatin in 250uL total volume is used per ChIP. Preloaded magnetic beads (Dynabeads M280 Streptavidin, Invitrogen #11205D) with anti-H3K9me3 (Diagenode, C15500003) or control-antibody were made by washing 68uL of beads with TBS/0.5% BSA 2x, followed by incubating with antibodies (1.8ug diluted in 105uL TBS/0.5% BSA) for 1 hr at 4C on a rotator. Excess streptavidin biotin-binding sites were blocked with 2x washes of 200uL of 5uM biotin (Sigma Aldrich, B4501) in TBS/0.5% BSA, rotating for 10 min at 4C. Beads were washed 2x with IP dilution buffer, then the chromatin added and incubated O/N at 4C on a rotator. The antibody/protein complexes were collected, washed several times and eluted in 1% SDS and 50mM NaHCO3. Crosslinks were reversed for 6hr at 65C in elution buffer with 200mM NaCl, treated with 1uL of 10ug/uL RNase A and 1uL of 20mg/mL Proteinase K. DNA was purified, the library was prepared (Illumina TruSeq Chip Library Prep Kit). Libraries were prepared using the Illumina TruSeq Chip Library Prep Kit. The resulting libraries were sequenced on the Illumina HiSeq 2000 or 2500 platform configured for 50bp single-end reads.
GEO Accession
GSM2610543