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. Lysates were prepared by resuspending crosslinked cells in lysis buffer (50 mM HEPES-KOH, (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% (vol/vol) glycerol, 0.5% (vol/vol) NP-40/Igepal CA-630 and 0.25% (vol/vol) Triton X-100, 2x Protease Inhibitors) and rotated for 10 min in the cold room. Nuclei were pelleted by centrifugation (2000xg, 5 min, 4??C) to pellet nuclei, which were resuspended in washing buffer (10 mM Tris-HCL (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA). Nuclei were pelleted  (2000xg, 5 min, 4??C) and resuspended in 5 ml digestion buffer (10 mM Tris pH 7.6, 5 mM MgCl2, 30 mM KCl, 3mM CaCl2, 2x Protease Inhibitors).   In-nuclei MNase digestion was started by addition of 1125U MNase (Worthington). Samples were then quickly vortexed and incubated at 37??C for 5 min while shaking at 500rpm. Reaction was quenched by addition of 10x Quenching buffer (150mM Tris pH8, 1000mM NaCl, 50 mM EDTA, 30 mM EGTA, 5% TritonX-100,5% N-Laroylsarcosine, 1% NaDoc) and by placing samples on ice. Samples were sonicated in Bioruptor Pico (Diagenode) to further promote DNA fragmentation. Insoluble material was removed by centrifugation at 20.000rpm for 10 min, 4??C and the soluble supernatant was used as ChIP Input. DNA fragment sizes of lysates were checked by agarose gel electrophoresis. Mainly mono-, di-, tri-nucleosomes were obtained.  M-280 Dynabeads (ThermoFisher) were precoated with Smarca4 antibody (Brg1, D1Q7F, Cell Signaling Technology #49360) or Chd4 antibody (D4B7, Cell Signaling Technology #12011) for 4 hours at 4 oC. Beads were washed, added to lysates and incubated over night at 4 oC while rotating. On the next day, beads were washed 4x with RIPA buffer (50 mM HEPES pH7.6, 100 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% NaDoc), 4x with RIPA containing 250 mM LiCl, and gently rinsed once with TE buffer+50 mM NaCl. Elution was carried out using SDS elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA, 1% SDS) at 65 oC for 30 minutes while shaking samples at 1500rpm. Samples were Proteinase K digested (0.2 mg/ml final) for 2 hours at 55 oC and crosslink was reversed over night at 65 oC. Samples were purified using Qiagen's PCR purification kit and used for ChIP-library preparation. 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 K36A
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-Smarca4/Brg1 (D1Q7F, Cell Siginput DNAling)