Mus musculus
Protocols: 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. 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 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).
ENA-FIRST-PUBLIC
2019-04-11T04:02:20Z
ENA-LAST-UPDATE
2018-06-15T11:55:41Z
External Id
SAMEA4730438
INSDC center name
EMBL
INSDC first public
2019-04-11T04:02:20Z
INSDC last update
2018-06-15T11:55:41Z
INSDC status
public
Submitter Id
E-MTAB-68221529059880:Input_H3.3ctrl-1_D0
broker name
ArrayExpress
cell line
129-B13
cell type
embryonic stem cell
common name
house mouse
genetic modification
gene knock out
genotype
H3f3a -/-
sample name
E-MTAB-68221529059880:Input_H3.3ctrl-1_D0
scientific_name
Mus musculus
time
0