ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A; K27A; K79A) and control mESCs
ChIP-seq for histone modifications and chromatin factors in H3.3 mutant (K9A; K27A; K79A) and control mESCs
Mouse embryonic stem cells (129XC57BL/6J) were cultured in ESC medium containing Knockout-DMEM (Thermo Fisher Scientific) supplemented with 15% EmbryoMax fetal bovine serum (ES-FBS Merck-Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression and Purification Facility at EMBL Heidelberg), 1% non-essential amino acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% Pen/Strep (Thermo Fisher Scientific), 1% of 55 mM beta-Mercaptoethanol solution (Merck Millipore). Upon thawing, cells were cultured on a layer of mouse embryonic fibroblasts (MEFs) and afterwards passaged on 0.1% gelatine-coated culture dishes, without feeder cells. Cells were maintained at 37°C with 5% CO2 and routinely tested for mycoplasma. The medium was changed daily, and cells were passed every two days. DNA for native ChIP was digested by MNase treatment to obtain mainly mono-nucleosomes using a modified protocol from Barski et al., 2007 (PMID: 17512414). For each IP, 20x106 cells were resuspended in digestion buffer (50 mM Tris-HCl pH 7.6; 1 mM CaCl2; 0.2% Triton-X), treated with 100 U MNase (Worthington) and incubated at 37°C for 5 minutes while shaking at 500 rpm. Samples were quickly moved to ice and MNase was quenched by addition of 5 mM EDTA (final). Lysates were sonicated for 3 cycles using Bioruptor Pico (Diagenode) and dialyzed against RIPA buffer for 3 hours at 4°C. Insoluble materials was pelleted at 10,000 rpm for 10 minutes at 4°C and supernatant was used as input for ChIP. To check the fragmentation pattern, input DNA was analyzed by agarose gel electrophoresis. A 5% fraction of input was set aside for sequencing. For native ChIP of histone modifications, Protein-G-Dynabeads (Invitrogen) were pre-coated with antibodies for 4 hours at 4°C. After overnight incubation of the pre-coated beads with chromatin lysates, beads were washed (3X with RIPA; 2X with RIPA + 300 mM NaCl; 2X with LiCl buffer-250 mM LiCl, 0.5% NP-40, 0.5% NaDoc) and finally rinsed with TE + 50 mM NaCl Buffer. Samples were eluted from ProteinG Dynabeads using SDS elution buffer (50 mM Tris-HCl ph 8.0; 10 mM EDTA; 1% SDS) at 65°C for 30 minutes with shaking at 1500 rpm. Proteinase K was added (0.2 µg/ml final) to the eluted samples and digestion was carried out for 2 hours at 55°C followed by QIAquick PCR purification (Qiagen). Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs) and, after Qubit quantification, the quality of individual libraries was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.