GSM8833391: ChIP,MEFwtlamin,rep1,Input; Mus musculus; ChIP-Seq
WT MEFs were grown to 90% confluence in DMEM (Gibco, 10566-016) supplemented with 10% (v/v) FBS (Gibco,10270106) and then fixed 10 min at RT with 1% formaldehyde (Sigma-Aldrich, F8775), adding 1:10 of formaldehyde solution (50 mM HEPES-KOH pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 11% formaldehyde) on cultured cells. Formaldehyde was then quenched with addition of Glycine (Carlo Erba, 453807) to a final concentration of 125 mM for 5 min at RT, followed by two washes with cold 1xPBS.  Cross-linked cells were rapidly collected in a falcon tube by scraping on ice and centrifuged at 2000 g at 4 °C.  Pellets of 3x106 cells were stored at -80 °C until sonication. Each 3x106 cell pellet was resuspended in 600 μl of cold Lysis Buffer: 50 mM HEPES-KOH, pH 7.5, 10 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100. After 10 min on a rotator at 4 °C, samples were centrifuged for 5 min at 1350g at 4 °C and resuspended in 130 μl of sonication buffer: 10 mM Tris-HCl pH 8.0, 2 mM EDTA, 1x Protease Inhibitor Cocktail (Roche, 04693116001), 1 mM PMSF (Sigma-Aldrich, 93482) and 0,1% SDS. Cells were subjected to lysis on ice for 1 h and homogenised by pipetting every 15 min. Total extracted chromatin was sonicated in a Covaris M220 focused-ultrasonicator using snap cap microTUBEs (Covaris, 520045) (water bath set to 7 °C, peak power 75.0, duty factor 10.0, cycles/burst 250, duration 420 s). Fragmentation of chromatin to an average size of 150-500bp was checked on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626). Samples were dilute to a final concentration 0.1% SDS by adding 1 volume of equilibration buffer: 10 mM Tris-HCl pH 8.0, 233 mM NaCl, 1.66% Triton X-100, 0.166% Deoxycholic acid sodium salt (DOC), 1 mM EDTA, 1x Protease Inhibitor Cocktail (Roche, 04693116001), 1 mM PMSF (Sigma-Aldrich, 93482and 0,1% SDS (for Lamin A/C). Samples were then centrifuged at 14000 g for 10 min at 4 °C to pellet insoluble material. Supernatants were quantified using Nanodrop 1000 spectrophotometer and 150 ug of chromatin was used for Lamin A/C immunoprecipitation in a final volume of 300 μl of IP buffer: 10 mM Tris-HCl pH8, 140 mM NaCl, 1.66% Triton X-100, 0.166% DOC, 0.1% SDS, 1 mM EDTA, 1x Protease Inhibitor Cocktail (Roche, 04693116001); 1 mM PMSF (Sigma-Aldrich, 93482). 3% of total chromatin was preserved at 4 °C as input normalization-control for each experimental condition. The remaining chromatin was incubated overnight on a rotator at 4 °C with 15 μl of Lamin A/C (Abcam. ab26300) antibodies. 20 μl of Protein G beads (Life Technology, 1004D) for each IP were washed twice in 0.1% BSA/IP buffer and incubated on rotator overnight at 4 °C. On the next day, protein G beads were added to each sample and incubated on rotator for 2 hours at 4 °C. Beads/IP complexes were then washed 10 min on rotator at 4 °C twice with IP buffer, twice with high-salt IP buffer: 10 mM Tris-HCl pH 8, 500 mM NaCl, 1.66% Triton X-100, 0.166% DOC, 0.1% SDS, 1 mM EDTA, 1x Protease Inhibitor Cocktail (Roche, 04693116001); 1 mM PMSF (Sigma-Aldrich, 93482); twice with RIPA-LiCl buffer: (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% DOC, 0.5% NP-40, 1x Protease Inhibitor Cocktail (Roche, 04693116001); 1 mM PMSF (Sigma-Aldrich, 93482); twice with 10 mM Tris-HCl pH 8.0. Crosslinking was reversed by incubating the beads and input at 65 °C overnight with 100 μl of Elution buffer: 10 mM Tris-HCl pH 8, 0,5 mM EDTA, 300 mM, and 0.4% SDS. The next day, all samples were diluted with 100 μl of 1x TE buffer, treated with 2.5 U of RNAse cocktail (Ambion, AM2286) at 37 °C for 120 min, followed by addition of 100 ug of Proteinase K (Invitrogen, AM2548) at 55 °C for 120 min. DNA was then isolated using phenol/chloroform (Sigma-Aldrich, 77617) extraction, followed by a back extraction of phenol/chloroform with additional volume of 1x TE buffer. DNA was precipitated in 2 volumes of cold ethanol, 0.3 M sodium acetate and 20 ug glycogen (Ambion AM9510) for overnight at -20 °C. Pellets were suspended in 31 μl of nuclease-free water and quantified using Qubit 2.0 fluorometer with Qubit dsDNA HS Assay Kits (Invitrogen, Q32854). The libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, BE7645L) and NEBNext Multiplex Oligos for Illumina (NEB, BE6440S). Libraries were then qualitatively and quantitatively checked and run on the TapeStation System