title: NextSeq 500 sequencing; ChIP-seq of H3.3K4A/K36A mutant ESCs 2
design: ChIP-seq of H3.3K4A/K36A mutant ESCs 2
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.  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:  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: organism
attributes.value: Mus musculus
attributes.tag: Experimental Factor: cell line
attributes.value: 129-B13
attributes.tag: Experimental Factor: genotype
attributes.value: H3f3a -/-; H3f3b K4A
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA