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Status |
Public on Oct 07, 2016 |
Title |
bio-Tet2-ChIP-in-Sall4KO-BirA |
Sample type |
SRA |
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Source name |
Sall4Δ/- (Sall4 KO) mES cells with Bio-tagged TET2
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Organism |
Mus musculus |
Characteristics |
strain background: 129P2/OlaHsd cell line: E14.1 cell type: embryonic stem cells genotype/variation: Sall4 delta/-, FlagBio-tagged TET2 knockin at Tet2 locus chip antibody: Dynabeads M-280 Streptavidin (Life Technologies)
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Growth protocol |
TT2 mouse ESCs were cultured on dishes coated with 0.1% gelatin (Millipore) without feeder cells. The medium contained Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, D9443), 10% Knockout Serum Replacement (KSR, Life Technologies), 1% dialyzed fetal bovine serum (FBS, Life Technologies), 3.5 mg/ml D-glucose (Amresco), 1× non-essential amino acids (Millipore), 1× β-mercaptoethanol (Millipore), 1,000 U/ml leukaemia inhibitory factor (LIF, Millipore), 105 μg/ml L-leucine (Sigma, L8912), 89 μg/ml L-arginine:HCl (for heavy medium, R10, Cambridge Isotope Laboratories, CNLM-539-0.5; for light medium, R0, Sigma, A8094) and 91 μg/ml L-lysine:HCl (for heavy medium, K8, Cambridge Isotope Laboratories, CNLM-291-0.5; for light medium, K0, Sigma, L8662).
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells for ChIP assays were crosslinked in 1% formaldehyde for 8-10 min at room temperature. The reaction was terminated by adding 2 M glycine to a final concentration of 125 mM. After being washed with PBS, the cells were resuspended in lysis buffer 1 (50 mM HEPES, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630 and 0.25% Triton X-100) and incubated for 10 min on ice. After centrifugation, the cells were resuspended in lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) and incubated for 10 min at room temperature. The chromatin fraction was resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% sodium N-lauroyl sarcosine) and sonicated into 200-400 base-pair (bp) fragments (Bioruptor, Diagenode). Chromatin fragments were immunoprecipitated overnight with antibodies against SALL1, SALL4A and TET1. The protein-antibody complexes were incubated with pre-washed Dynabeads Protein A or G (Life Technologies) for 1 hr. The beads were sequentially washed with the following buffers: once with FA-lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM HEPES, pH 7.9, 2 mM EDTA and 0.5% sodium deoxycholate), twice with FA high salt buffer (500 mM NaCl, 1% Triton X-100, 50 mM HEPES, pH 7.9, 2 mM EDTA and 0.5% sodium deoxycholate), once with LiCl buffer (250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0) and twice with TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA). Protein complexes were eluted by incubation with 250 μl of freshly prepared elution buffer (1% SDS and 100 mM sodium bicarbonate) for 15 min at room temperature. The elution step was repeated once, and the eluted fractions were combined. For ChIP of bio-SALL4A, chromatin fragments were incubated with Dynabeads M-280 Streptavidin (Life Technologies) equilibrated with PBS + 1% BSA. Triton X-100 was added to a final concentration of 1%. Protein-bound beads were washed sequentially with following buffers: 2% SDS twice, FA high salt buffer twice, LiCl buffer (250 mM LiCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl, pH 8.0) once and TE buffer twice. Protein complexes were eluted by incubating the beads in 300 μl of elution buffer at 65℃ with agitation at 800 rpm overnight. After crosslinking reversal and RNase A and proteinase K treatment, DNA was extracted with phenol-chloroform and precipitated with ethanol. For DNA immunoprecipitation assays, genomic DNA was isolated by conventional phenol extraction and proteinase K treatment. The purified genomic DNA was sonicated into 200-400 bp fragments. Adapter-ligated DNA libraries were constructed. DNA fragments were denatured in 0.4 M NaOH by incubation at 95℃ for 10 min and then placed on ice immediately and neutralised by adding an equal volume of 2 M ammonium acetate. Samples (3 μg) of the adapter-ligated DNA libraries were immunoprecipitated with 5 μg of mC or hmC antibodies, and 5 μg of DNA with 0.2 μl of caC anti-serum. For mRNA deep sequencing analysis, total RNA was isolated by TRIzol Reagent (Life Technologies), and mRNA with a poly (A) tail was purified and subjected to sequencing library construction. The ChIP-seq libraries were constructed using NEBNext DNA Sample Prep Master Mix (NEB).DNA libraries were subjected to size-selection using AMPure XP beads (Beckman Coulter) and PCR amplification (18-20 cycles for ChIP-seq DNA libraries and 12-16 cycles for DIP-seq libraries).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 4000 |
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Description |
TET2 bio-ChIP in Sall4Δ/- (Sall4 KO) mES cells with Bio-tagged TET2
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Data processing |
Basecalls performed by BGI, Shenzhen ChIP-seq reads were aligned to the mm9 genome assembly using bowtie version 0.12.7 with only unique mapped reads were kept Data were filtered using samtools to remove potential PCR duplicates peaks were called using MACS (v1.4.2) RNA-Seq data were mapped by TopHat and quantitified by Crosslinks. Genome_build: mm9 Supplementary_files_format_and_content: wig files were generated using IGV tools with fragment extension 300 bp
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Submission date |
Feb 18, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Zhuqiang Zhang |
E-mail(s) |
zhangzhuqiang@ibp.ac.cn
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Organization name |
Institute of Biophysics, CAS
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Street address |
15 Datun Road, Chaoyang District.
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City |
Beijing |
State/province |
--- |
ZIP/Postal code |
100101 |
Country |
China |
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Platform ID |
GPL21103 |
Series (1) |
GSE57700 |
Cooperative Action Between SALL4A and TET Proteins in Stepwise Oxidation of 5-Methylcytosine |
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Relations |
SRA |
SRX1591000 |
BioSample |
SAMN04502949 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2065712_s4ko-BirA-bioTet2.bigwig |
226.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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