title: Illumina HiSeq 2000 sequencing; ChIP-seq of H3.3K4A/K36A mutant ESCs and neurons
design: ChIP-seq of H3.3K4A/K36A mutant ESCs and neurons
library_construction_protocol: Mouse embryonic stem cells (129B-13) were cultured in ESC media containing Knockout-DMEM (Thermo Fisher) with 15% EmbryoMax FBS (Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression Facility at EMBL Heidelberg), 1% non-essential amino acids, 1% Glutamax, 1% Pen/Strep, 1% of 55mM beta-Mercaptoethanol solution. Cells were maintained at 37C with 5% CO2. Fresh cell pellets of 20 Mio. cells were resuspended in 20 ml of 1% fresh paraformaldeyde (PFA) diluted in PBS and rotated for 10 min at room temperature. Unreacted PFA was quenched with glycine. Cells were washed twice to remove PFA, snap-frozen and stored at -80C.  Frozen cell pellet of 10 Mio. cells was resuspended in digestion buffer, 100 U MNase (Worthington) were added and samples were incubated at 37C for 5 minutes while shaking at 500 rpm. Samples were immediately moved to ice and MNase was quenched by addition of EDTA. Lysates were sonicated using Bioruptor Pico (Diagenode) and dialyzed against RIPA buffer for 3 hours at 4C. Insoluble material was pelleted at 10.000rpm for 10 min, 4C  and supernatant was used as input for ChIP. To check that mostly mono-nucleosomes and di-nucleosomes were obtained, DNA fragment sizes of inputs were analyzed by agarose gel electrophoresis.   For native ChIP of histone modifications, Protein-G-Dynabeads (Invitrogen) were precoated with antibodies for 4 hours at 4C while rotating. We used the following antibodies: H3K4me1 (39297, active motif), H3K4me3 (39915, Active Motif), H3K27ac (39685, Active Motif). Beads were added to lysates and rotated over night at 4C. On the next day, beads were washed with 3x RIPA, 2xRIPA+NaCl, 2x LiCl Buffer and finally rinsed 1x with TE+50mM NaCl Buffer. Samples were eluted from ProteinG Dynabeads using SDS-elution buffer (50 mM Tris-HCl ph8.0, 10 mM EDTA, 1% SDS) at 65C for 30 minutes, shaking at 1500 rpm.  Proteinase K was added (0.2 mg/ml final) to eluted samples and digestion was carried out for 2 hours at 55C followed by PCR purification (Qiagen). Samples were eluted from ProteinG Dynabeads using SDS-elution buffer (50 mM Tris-HCl ph8.0, 10 mM EDTA, 1% SDS) at 65C for 30 minutes, shaking at 1500 rpm.  Proteinase K was added (0.2 mg/ml final) to eluted samples and digestion was carried out for 2 hours at 55C followed by PCR purification (Qiagen). For Inputs (MNase-Seq) 300 ng of prepared MNase-digested DNA was used for library preparation. For ChIP, entire IP-eluate was used for library preparation.  Sequencing libraries were prepared using DNA Ultra II library preparation kit (NEB) according to the manufacturer???s instructions.  Number of Amplification cycles was adjusted depending on ChIP:  H3K27ac 10x cycles, H3K4me3 10x cycles, H3K4me1 11x cycles, H3.3 12x cycles, H3K36me3 12x cycles, Inputs 10x cycles. Samples were barcoded, pooled and sequenced on Illumina???s HiSeq2000 Sequencer (50 bp single-end mode) or NextSeq 500 Sequencer (75 bp single-end mode).
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: H3K4me3 (39915, active motif)
attributes.tag: Experimental Factor: time
attributes.value: 0
attributes.tag: Experimental Factor: genotype
attributes.value: H3f3a -/-