GSM4395464: TSB564_BJ_Senescent_anti-H4R3me2sym_shNS; Homo sapiens; ChIP-Seq
Native ChIP was performed as described in Wagschal et al., 2007 (Wagschal et al., 2007), summarized as follows, noting alterations to the original protocol.  5-10 million BJ fibroblasts, either senescent or quiescent via contact inhibition, were harvested via trypsinization, making sure that cells are in a single cell suspension.  Cells were then transferred to a 15ml conical tube and pelleted at 3000g, for 5 minutes, at 4C.  All subsequent steps and buffers, unless otherwise noted, were performed and maintained at 4C.  Cells were resuspended in 2ml nuclei preparation buffer I (0.3M sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 15mM Tris-HCl ph 7.5, 0.5mM DTT, 1x dissolved cOmplete Protease Inhibitor Cocktail), and were then mixed gently with 2ml of nuclei preparation buffer 2 (0.3M sucrose, 60mM KCl, 15mM NaCl, 5mM MgCl2, 0.1mM EGTA, 15mM Tris-HCl ph 7.5, 0.5mM DTT, 1x dissolved cOmplete Protease Inhibitor Cocktail, 0.4% NP-40), and the mixture was then incubated on ice for a maximum of 5 minutes.  8 ml of nuclei preparation buffer III was added to 2 new separate 14ml polypropylene tubes.  2ml of the 4ml cell suspension was layered on top of each 8ml sucrose cushion and centrifuged in a pre-chilled swing-out rotor at 10,000g, for 20 minutes at 4C.  The supernatant was removed from the top, with attention paid to ensure that none of the supernatant remained.  The nuclear pellet was resuspended in 1ml of MNase digestion buffer (0.32M sucrose, 4mM MgCl2, 1mM CaCl2, 50mM Tris-HCl ph 7.5, 0.1 mM PMSF), split into two aliquots of 500ul in two separate 1.5ml Eppendorf tubes, 1ul of MNase enzyme (NEB-M0247S) was added to each tube, and the tubes were incubated for 2 and 5 minutes at 37C.  The reaction was stopped by adding 5ul of 0.5M EDTA and 5ul of 0.5M EGTA to each reaction, which were then chilled on ice.  Each tube was centrifuged for 10,000 rpm for 10 minutes at 4C and the supernatant, which contains the first fraction of soluble chromatin, was collected.   Each remaining nuclear pellet was then resuspended in 500ul dialysis-lysis buffer (1mM Tris-HCl ph 7.5, 0.2 mM PMSF, 0.2mM EDTA), incubated for 1 hour at 4C, and samples were re-centrifuged at 10,000 rpm for 10 minutes at 4C and each samples' supernatant, containing the second fraction of soluble chromatin, was collected.  Concentrations of each fraction were then measured via Qubit, and the fragment size distribute of each fraction was checked via agarose gel electrophoresis to confirm similar digestion between each biological sample.  Fractions were then diluted in their respective buffers to ensure a similar concentration of chromatin in each fraction, and then equal volumes of each fraction of each biological sample were pooled and diluted so that each ChIP reaction contained ~10ug chromatin in 500ul of buffer, and was equal parts MNase digestion buffer and dialysis-lysis buffer, and equally parts each fraction of chromatin.  1ml of ChIP incubation buffer (50mM Tris-HCl, ph 7.5, 5mM EDTA, 50mM NaCl, 1x dissolved cOmplete Protease Inhibitor Cocktail) was then added to each 500ul ChIP reaction.  5ug of antibody was then added to each reaction, which were then rotated overnight at 4C.  20 uL of Protein G Dynabeads (Thermo Fisher) per ChIP reaction were then washed and blocked overnight in 1% BSA/glycogen in ChIP incubation buffer, and resuspended in their original volume of ChIP incubation buffer.      After the reactions' overnight incubation, 20ul of the beads were then added to each ChIP reaction and incubated under rotation for 4 hours at 4C.  Beads were then washed, using the same magnetic washing protocol as with the fixed ChIP reactions, 2 times with Washing Buffer A (50mM Tris-HCl, ph 7.5, 10mM EDTA, 75mM NaCl), 2 times with Washing Buffer B (50mM Tris-HCl, ph 7.5, 10mM EDTA, 125mM NaCl), and 2 times with Washing Buffer C (50mM Tris-HCl, ph 7.5, 10mM EDTA, 175mM NaCl).  Beads were then resuspended in 48 ul Proteinase K digestion buffer with 2 mg/ml Proteinase K, and incubated for 30 minutes at 50C, after which the supernatant was collected and purified via QIAquick PCR Purification Kit (QIAGEN), and were eluted in EB.  Samples were then further sonicated for 10 minutes to ensure that immunoprecipitated chromatin from polynucleosomes would between 100-300bp for sequencing. Libraries were then prepared for deep sequencing with the Illumina HiSeq 2500 system using the KAPA LTP Library Preparation Kit (KK8232), following manufacturer's instructions.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.