Sc7292x

ChIP was done as described [14]. Cells (5×10⁶ cells per ChIP) were cross-linked with 1% formaldehyde and lysed in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 1% SDS, 0.1% sodium deoxycholate, 1 mM PMSF, protease inhibitors). To generate 200–500 bp DNA fragments, cells were sonicated 4 × 10 min in a Bioruptor (Diagenode). After sedimentation, the supernatant was diluted 10x in RIPA and incubated with anti-lamin A/C (10 µg, sc7292x, Santa Cruz Biotechnology) or lamin B1 (10 µg, ab16048, Abcam) antibodies coupled to Dynabeads Protein G (Invitrogen). ChIP samples were washed 3x in ice-cold-RIPA. Samples were incubated for 6 h at 68°C in elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% SDS) and 40 ng proteinase K for crosslink reversal and DNA elution. DNA was extracted and libraries prepared and sequenced on an Illumina HiSeq2500.

Proteins were separated by 10% SDS-PAGE, transferred onto an Immobilon-FL membrane (Millipore) and membranes blocked with Odyssey blocking buffer (LI-COR). Membranes were incubated in Odyssey blocking buffer with antibodies against lamin A/C (1:2000, sc7292x, Santa Cruz Biotechnology; or a rabbit anti-lamin A/C antibody [44]; 1:1000), pre-lamin A (1:200, sc6214, Santa Cruz Biotechnology), lamin B1 (1:1000, sc6216, Santa Cruz Biotechnology), ZMPSTE24 (1:1000, AP2415a, Abgent) or γ-tubulin (1:10,000, T5326, Sigma-Aldrich). Proteins were detected with IRDye-800- or IRD IRDye-680-coupled secondary antibodies.

Ten million cells were harvested in 1X PBS and lysed in 1 ml ice-cold IP buffer (20 mM Tris-HCL p^H8, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA and 1X protease inhibitor cocktail) for 30 min with constant mixing at 4 °C. Following centrifugation at 12,000 rpm for 20 min at 4 °C, the soluble supernatant fraction was pre-cleared with Protein A sepharose beads for 1 h at 4 °C. Five percent of the lysate was kept as “Input” to serve as a positive control. Lysates were then mixed with 60 μl of Protein A sepharose beads that were pre-incubated with 4 μg of H4/H2AS1ph (ab177309, Abcam), Lamin A/C (sc-7292 X, Santa Cruz) or IgG (Biogenesis 5180-2104) antibodies for 4 h and blocked in salmon sperm DNA for 40 min. Following overnight incubation with constant agitation at 4 °C, the antibody-beads-protein complexes were centrifuged and washed three times with low salt buffer (10 mM Tris-HCL p^H7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100 and 1X protease inhibitor cocktail) and IP samples were eluted in 2X Laemmli buffer at 95 °C for 10 min.