title: GSM1155629: Heterotypic CenH3 (CenH3/H3) ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells are harvested by a low speed centrifugation (500g, 5min, 4°C), washed twice in cold PBS and resuspended in three cell volumes of cold NB buffer (15mM Tris HCl pH 7,5, 15mM NaCl, 60mM KCl, 300mM Sucrose, 1X complete EDTA free (roche diagnostic), 25ng/ml trichostatin A, 10mM beta glycerophosphate, 5mM sodium fluoride, 0,2mM sodium orthovanadate). Four pellet volumes of NB buffer containing 1% NP40 is then added to the cell suspension and incubated 10 min at 4°C on a wheel. After centrifugation at 1200g for 5 min at 4°C nuclei are washed with 3 nuclei volumes of NB buffer and resuspended in ½ nuclei volume of NB buffer. After adding CaCl2 to a final concentration of 2mM nuclei are partially digested with MNase (Nuclease S7 roche diagnostic) to give a range of oligonucleosome. Nuclei are centrifuged 5 min at 16 000g 4°C and the supernatant is dialyzed at least 3h against 1L of lies buffer (Tris HCl pH 7,5 1mM, EDTA pH 8 0,2mM, 1X complete EDTA free (roche diagnostic), 25ng/ml trichostatin A, 10mM beta glycerophosphate, 5mM sodium fluoride, 0,2mM sodium orthovanadate). To extract as much chromatin as possible from the remaining pellet, it is resuspended in one pellet volume of Lysis buffer and dialyzed for at least 3h against 1L of dialysis buffer. After centrifugation 16 000g 4°C 5 min the supernatant of the resuspended pellet is mixed with the first supernatant. The extract is loaded on top of a 5-30% sucrose gradient made in the lies buffer and centrifuged for 16h 29000rpm at 4°C in the SW32 rotor (Beckman Coulter). 1ml fractions are made and 50l is analyzed by agarose gels to determine the nucleosome composition of each fractions. After increasing NaCl concentration to 300mM, Tris/HCl pH 8 to 10mM and NP40 to 0,1%, fractions containing mononucleosomes are pooled and histone are immunoprecipitated with Flag M2 Agarose beads (Sigma Aldrich) for 4h at 4°C or with protein A/G magnetic beads (Thermo scientific) coated with CENP-A (3-19) antibody (Enzo life sciences) overnight at 4°C on a wheel. Beads are washed 3 times with the IP buffer (20mM Tris HCl pH7,5, 300mM NaCl, 0,2mM EDTA pH 8, 0,1% NP40, glycerol 10%, 1X complete EDTA free (roche diagnostic), 25ng/ml trichostatin A, 10mM beta glycerophosphate, 5mM sodium fluoride, 0,2mM sodium orthovanadate) and elute 2 times 1h at 4°C for the flag IP with ½ beads volumes of elution buffer (IP buffer containing 0,4g/ml flag peptide, Sigma Aldrich)  2 times 3h at 4°C for the CENP-A IP with ½ beads volumes of elution buffer (IP buffer containing 0,5g/ml CENP-A 3-19 peptide). Libraries were prepared according to standard Illumina protocols for paired-end sequencing on the HiSeq2000 machine
attributes.tag: GEO Accession
attributes.value: GSM1155629