GSM1541019: Hela_digest_LMNB1; Homo sapiens; ChIP-Seq
Chromatin preparation by micrococcal nuclease (MNase) digestion: For chromatin preparation by MNase, cells were fixed with 1% formaldehyde for 10 min, washed and formaldehyde quenched with 125 mM glycine for 5 min. After a wash in PBS, cells were scrapped in cold PBS containing 0.5 mM PMSF, sedimented and suspended in hypotonic buffer (10 mM Tris pH 7.5, 5 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, protease inhibitors) for 30 min on ice before Dounce homogenization (50 strokes). MgCl2 was adjusted to 5 mM and NP-40 added to 0.1%. The lysate was kept on ice for 10 min before centrifugation at 2400 g for 10 min. The pellet was resuspended in MNase buffer (20 mM Tris pH 7.5, 15 mM NaCl, 1 mM CaCl2) at 25x106 cells/ml and pre-incubated at 37oC for 5 min before adding 0.37 U MNase (Sigma; N5386) per 106 cells. Incubation was at 37°C for 5 min and digestion stopped with 10 mM EDTA on ice for 10 min. Samples were diluted to 8x10E6 cells/ml with lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS, 0.5 mM PMSF and 1x protease inhibitors). Release of digested chromatin and lamins was facilitated by pulse-sonication with a 3 mm probe for 6 times 10 sec (1 sec on/off) at 20% amplitude with cooling on ice between each round (see Results). Samples were centrifuged at 10,000 g for 10 min at 4°C and the supernatants (chromatin fraction) collected.  Chromatin was then diluted 5x in NP-40 buffer (20 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1x protease inhibitors) without SDS to decrease SDS concentration to 0.1%. Chromatin was pre-cleared for 1 h at 4oC with Protein A/G agarose beads (Santa Cruz sc-2003) and incubated overnight at 4oC with the same anti-lamin A/C antibody as above (6 µg for 6x106 cells) or with anti-lamin B1 antibodies (Abcam ab16048; 3 µg for 6x10E6 cells). Immune complexes were collected using Protein A/G agarose beads for 2 h on a rotator at 4°C, washed 6x 5 min on a rotator at 4°C as follows: once in 20 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitors; twice in 20 mM Tris pH 8.0, 5 mM EDTA, 500 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitors; twice in 10 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholate, protease inhibitors; and once in TE buffer. Note that the NaCl- and LiCl-based washes used here are recommended in ChIP protocols using agarose beads. Crosslink was reversed and DNA was eluted and purified as above. Library preparation and sequencing were done as above. The sequencing library was prepared according to the Illumina protocol for the HSeq2500 at the Norwegian Sequencing Center.