GSM2610549: LNCaP Lamin A/C ChIP-seq; Homo sapiens; ChIP-Seq
Cells (10^7 per ChIP) were cross-linked in suspension for 10 min in PBS containing 1% formaldehyde before quenching with 1.25mM glycine. Pellets were collected and resuspended in hypertonic buffer (10mM Tris pH 7.5, 5mM NaCl, 1.5mM MgCl2, 0.5mM DTT, 0.5mM PMSF protease inhibitor), incubated on ice for 30 mins, and dounced 50x. MgCl2 was then adjusted to 5mM, NP-40 (IGEPAL) was added to final concentration of 0.1%, incubated on ice for 10 mins, before centrifugation at 2400g for 10 mins. The pellet was resuspended in MNase buffer (20mM Tris pH 7.5, 15mM NaCl, 1mM CaCl2) at 25 million cells/ml, incubated for 5 mins at 37°C, 0.5U MNase was added per 1 million cells and then incubated at 37°C for 5 mins.  Digestion was stopped with 10mM final EDTA in ice for 10 mins. Samples were diluted to 8 million cells/ml with lysis buffer (50mM Tris pH8, 10mM EDTA, 0.5% SDS, 0.5mM PMSF and prostease inhibitors)  before sonication. The sonicated lysate was spun at 16,100g for 10 mins at 4°C and aliquoted. The chromatin was diluted 5x in NP-40 buffer (20mM Tris pH 8, 5mM EDTA, 150mM NaCl, 1% NP-40 and protease inhibitors, SDS 0.1%), pre-cleared with Santa Cruz A/G beads for 1 hr at 4°C, spun at 1000rpm for 1 min at 4°C, before adding antibody 5ug lamin B1 (Abcam, #ab16048) and 10ug lamin A/C (Santa Cruz. #sc7292) for overnight incubation with rotation. ChIP material was collected and washed 3x in 1 ml ice-cold RIPA buffer. Crosslink was reversed and DNA eluted for 6 h on a shaker at 37°C in elution buffer (50mM NaCl, 20mM Tris-HCl, pH 7.5, 5mM EDTA, 1% SDS) containing 0.5μg/ml RNase A and 2μg/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.