title: GSM2805185: EZH2 ChIP mES cell replicate 1; Mus musculus; ChIP-Seq
library_construction_protocol: mESC were MEF depleted, crosslinked with 1% formaldehyde in PBS for 10min in conical tubes while gently rotating at RT and quenched by adding Glycine to a final concentration of 0.125M and left at RT for 5min on rotator. Crosslinked cells were spun down for 3min at 3500 rpm (2850 rcf) and lysed in cell lysis buffer  (50mM Tris PH 8.0, 50mM NaCl, 1mM EDTA, 5mM MgCl2, 0.25% NP-40, Roche complete EDTA-free protease inhibitor tablet(PI)* 0.2% PMSF* and 0.2% DTT*(*freshly-added)) at 2-2.5x107 cells/ml. Cells were left on ice for 10 min and dounced 10x using the tight pestle. Nuclei was spun down at 2850 rcf in 15ml conical tubes and the supernatnant was completely removed and nuclei was resuspended in sonication buffer (20mM Tris PH 8.0, 1% SDS, 2mM EDTA, 0.5mM EGTA, 0.5mM PMSF*, PI*(freshly added) at 5.0x107 nuclei/ml. Resuspended nuclei was left on ice for 10min and sonicated using the Bioruptor sonicator, at high setting 30’’ on 45’’ off twice for 15min. Water and ice was changed in between each sonication. IP was performed with using rabbit anti EZH2 (CST,D2C9), 1-2.5x106 nuclei were used per IP reaction, by adding 20-50 µl of sonicated chromatin to 1-1.25ml of IP buffer (20mM Tris PH 8.0, 0.5% Triton X-100, 150mM NaCl, 10% glycerol, 2mM EDTA) with the respective antibodies and after 4 hours of incubation protein A (40-50 µl) was added to the IP reaction and incubated for 4 more hours. Each wash was done on the rotator at RT for 5min as follows: x1 low salt wash (20mM Tris PH 7.8, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl), x3 high salt wash (20mM Tris PH 7.8, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl), x1 LiCl wash (20mM Tris PH 7.8, 1% NP-40, 1% NaDeoxycholate, 2mM EDTA, 250mM LiCl), x2 TE wash.Elution was done on the IPed samples in 2x250µl of elution buffer (1% SDS, 0.1M NaHCO3) on the rotator at RT. To the 500µl elution buffer, 20 µl of 5M NaCl was added and samples were incubated in a thrmomixer for 4hr-O/N at 65oC to reverse the crosslink (same was done for the Input material). Next, 15µl of 1M Tris PH 7.8, 1-2 µl glycoblue (Ambion) and 2 µl of proteinase K was added to the reaction mix and incubated at 65oC for 30-60min. DNA was extracted from the reversed crosslink material using phenol/chloroform extraction and EtOH precipitation. Library constructing for sequencing was prepared as described elsewhere (Bowman et al., BMC Genomics 2013).
attributes.tag: GEO Accession
attributes.value: GSM2805185