NextSeq 500 sequencing; ChIP-seq of H3.3K4A/K36A mutant ESCs 2
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
ENA first public
2020-01-28
ENA last update
2019-12-13
ENA-CHECKLIST
ERC000011
External Id
SAMEA6415111
INSDC center alias
EMBL
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2020-01-28T17:04:30Z
INSDC last update
2019-12-13T13:35:11Z
INSDC status
public
Submitter Id
E-MTAB-8614:ChIP_CHD4_s6_K36A2
broker name
ArrayExpress
cell line
129-B13
cell type
embryonic stem cell
common name
house mouse
genetic modification
gene knock out
genotype
H3f3a -/-; H3f3b K36A
sample name
E-MTAB-8614:ChIP_CHD4_s6_K36A2
Sequenced DNA Library
library_name
ChIP_CHD4_s6_K36A2_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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).