title: ChIP_H3K36me3_H3.3K36A-1_D12
organism: Mus musculus
description: Protocols: mESC growth: 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. mESCs were used as neuronal differentiation Day 0 (D0). Neuronal Differentiation: mESCs were differentiated into glutamatergic neurons according to Bibel et al. (Nature Protocols, 2007) with small modifications. mESCs were resuspended in differentiation media without LIF and grown in suspension using non-adherent plates to promote the formation of embryoid bodies. Differentiation media was changed every two days. On day 4 retinoic acid was added to the media. Embryoid bodies were cultured for 4 additional days in the presence of retinoic acid to obtain neural precursor cells on differentiation day 8. For neural monolayer culture, plates were pre-coated with Poly-D-Lysin and Laminin. On day 8, Embryoid bodies were dissociated with trypsin and cells were plated in N2 media (regular DMEM supplemented with 1xN2 and 1xB27-VitaminA (Thermo Fisher)) at a density of 200.000 cells/cm2 on precoated plates. N2 media was changed every two days without exposing neuron culture to oxygen. Four days after plating, neurons were harvested after a total of 12 differentiation days (D12). Lysates were prepared by resuspending crosslinked cells in hypotonic buffer (15 mM HEPES pH8, 5 mM MgCl2, 10 mM KCl, 0.5mM EDTA, 2x Protease Inhibitors) followed by douncing to release intact nuclei. Nuclei were pelleted and resuspended in digestion buffer (15 mM HEPES pH8, 5 mM MgCl2, 30 mM KCl, 3mM CaCl2, 2x Protease Inhibitors) by douncing. In-nuclei MNase digestion was started by addition of 150U MNase (Worthington). Samples were then quickly vortexed and incubated at 37C for 5 min while shaking at 500rpm. Reaction was quenched by addition of 10x Quenching buffer (150mM HEPES pH8, 1500mM NaCl, 50 mM EDTA, 30 mM EGTA, 0.2% TritonX-100) and by placing samples on ice. 0.1% sodium deoxycholate, 0.5% sarcosine and 1% Triton-X were added to lysates and samples were sonicated in Bioruptor Pico (Diagenode) to further promote DNA fragmentation. Insoluble material was removed by centrifugation at 20.000rpm for 10 min, 4C 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. Protein-G Dynabeads were precoated with H3.3 antibody (Millipore) for 4 hours at 4C. Beads were washed, added to lysates and incubated over night at 4C while rotating. On the next day, beads were washed 8x with RIPA buffer (50 mM HEPES pH7.6, 100 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Sodium deoxycholate) 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 65C for 30 minutes while shaking samples at 1500rpm. Samples were Proteinase K digested (0.2 mg/ml final) for 2 hours at 55C and crosslink was reversed over night at 65C. 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: 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: ENA-FIRST-PUBLIC
attributes.value: 2019-04-11T04:02:19Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-05-16T11:07:46Z
attributes.tag: External Id
attributes.value: SAMEA4666005
attributes.tag: INSDC center name
attributes.value: EMBL
attributes.tag: INSDC first public
attributes.value: 2019-04-11T04:02:19Z
attributes.tag: INSDC last update
attributes.value: 2018-05-16T11:07:46Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6822:ChIP_H3K36me3_H3.3K36A-1_D12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 129-B13
attributes.tag: cell type
attributes.value: neuron
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genetic modification
attributes.value: induced mutation
attributes.tag: genotype
attributes.value: H3f3a -/-; H3f3b K36A
attributes.tag: sample name
attributes.value: E-MTAB-6822:ChIP_H3K36me3_H3.3K36A-1_D12
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: time
attributes.value: 12