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| Status |
Public on Mar 27, 2012 |
| Title |
mES_Dnmt-TKO_H3K27m3_ChIP-BS |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
origin: Embryonic stem cells
technique: ChIP-bisulfite sequencing
antibody: H3K27me3
antibody vendor: Millipore
antibody catalog #: 07-449
antibody lot #: DAM-1588246
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| Growth protocol |
HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all Gibco/Invitrogen) at 37 °C in 5% CO2 atmosphere.
ES cells were cultured in normal cell culture containing ES-grade serum supplemented with LIF. These cells were grown on gelatin-coated plates
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 0.125 M glycine and washed at 4C with three buffers: (i) PBS, (ii) buffer of composition 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6 and (iii) 0.15 M NaCl in HEG buffer (1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6). Cells were then suspended in ChIP incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, HEG) and sheared using a Biouptor (Diagenode). Sonicated chromatin was centrifuged for 5 min and then incubated overnight with 2 ug of antibody and protein A/G beads (Santa Cruz). Beads were washed six times with different buffers at 4C: twice with solution of composition 0.1% SDS, 0.1% DOC, 1% Triton, 150 mM NaCl, HEG, once with the solution same as before but with 500 mM NaCl, once with solution of composition 0.25 M LiCl, 0.5% DOC, 0.5% NP-40, HEG and twice with HEG. Precipitated chromatin was eluted with 400 µl of elution buffer (1% SDS, 0.1 M NaHCO3), incubated at 65C for 4 h in the presence of 200 mM NaCl, phenol extracted and precipitated with 20 ug of glycogen at -20C overnight.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
100 ng ChIP DNA or 10 ng MethylCap DNA was first subjected to end-repair in a 30 µl reaction containing 6 units T4 DNA polymerase, 2.5 units DNA Polymerase I (Large Klenow Fragment), 20 units T4 Polynucleotide Kinase (all New England Biolabs), dATP, dCTP, dGTP and dTTP (0.125 mM each), and 1x T4 Ligase buffer with ATP for 30 min at 20C. Illumina sequencing generates sequences corresponding to the 5'-ends of the input DNA fragments. Therefore, fill-in of 5'-overhangs did not alter sequenced DNA methylation patterns. Purification was performed using a standard phenol:chloroform:isoamyl alcohol (25:24:1) protocol and ethanol precipitation. DNA was then adenylated in a 20 µl reaction containing 10 units Klenow Fragment (3'->5' exo-) (New England Biolabs), 0.5 mM dATP and 1x NEB buffer 2 for 30 min at 37C. After phenol extraction and ethanol precipitation, Illumina genomic DNA adapters containing 5-methylcytosine instead of cytosine (atdbio, Southampton), preventing deamination during bisulfite conversion, were ligated. In a 20 µl reaction, DNA was incubated with 1.5 µM preannealed adapters, 2000 units T4 DNA Ligase (NEB) and 1x T4 Ligase buffer with ATP, for 16-20 h at 16C. Adapter-ligated DNA fragments were subsequently purified by phenol extraction and ethanol precipitation, and size-selected on gel. 50 ng sheared and dephosphorylated E-coli K12 genomic DNA was added to adapter-ligated DNA as carrier during size-selection and bisulfite conversion. DNA was run on 2.5% Nusieve 3:1 Agarose (Lonza) gels. Lanes containing marker (50 bp ladder New England Biolabs) were stained with SYBR Green (Invitrogen), and size regions to be excised were marked with toothpicks. To obtain 80-280 bp (low) and 280-430 bp (high) insert sizes, adapter-ligated DNA fragments from 200-400 and 400-550 bp, respectively, were excised. The Illumina adapters cause the fragments to run slower, presumably due to the forked structure. Note that after PCR, dsDNA libraries appear at 140-340 bp and 340-490 bp, in accordance with exact sizes. DNA was isolated from gel using the MinElute Gel Extraction kit (QIAGEN). The low and high libraries were kept separate in subsequent steps. Before proceeding to bisulfite conversion, analytical PCRs were performed to check the libraries. Amplifications were performed in 10 µl reactions containing 0.3 µl template DNA (from 20 µl eluted after size-selection), 0.5 units Pfu Turbo Cx Hotstart DNA Polymerase (Stratagene), Illumina primers LPX 1.1 and 2.1 (0.3 µM each), dNTPs (0.25 mM each), 1x Turbo Cx buffer, under the following thermocycler conditions: 94C for 5 min, n x (94C for 30 s, 65C for 30 s, 72C for 1 min) and 72C for 7 min. We tested three different cycle numbers (n) - 10, 15 and 20 cycles - and analyzed PCR products on 4-20% TBE Criterion precast gels (BioRad) and subsequent SYBR Green staining. Adapter-ligated and size-selected DNA was subjected to two subsequent 5h bisulfite treatments using the EpiTect Bisulfite kit (QIAGEN) following the manufacturer's protocol for DNA isolated from FFPE tissue samples. After bisulfite conversion, analytical PCRs were performed as before to determine the minimum number of cycles required in the final amplification step for each sample. In this case, tested cycle numbers were several cycles higher as before bisulfite conversion, and 15, 19 and 22 PCR cycles were performed. The minimum cycle number for final large-scale amplification was determined as the lowest cycle number that generated enough PCR product of the desired size range to be visualized on analytical gels as above. Large-scale amplification was performed by 8 reactions of 25 µl, containing 3 µl DNA (from 40 µl eluted after bisulfite conversion, remains were stored at -80C as back-up), 1.25 units Pfu Turbo Cx Hotstart DNA Polymerase (Stratagene), primer LPX 1.1 and 2.1 (0.3 µM each), dNTPs (0.25 mM each), 1x Turbo Cx buffer and thermocycler conditions as above. Amplified libraries were purified with the MinElute PCR Purification kit (QIAGEN), and subsequently purified from gel essentially as described above; whole gels were stained with SYBR Green and no carrier DNA was added. Final libraries were analysed on analytical 4-20% TBE Criterion precast gels (BioRad), and measured by Quant-iT dsDNA HS Assays (Invitrogen). The protocol for preparation of captured DNA for bisulfite deep sequencing was adapted from 27-29. Sequence reads were generated on the Illumina Genome Analyzer IIx using a standard 36-base protocol. After sequencing low and high library reads were combined.
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| Data processing |
Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina HiSeq2000 platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Mapping of bisulfite-converted sequence reads was done using a custom-made pipeline. To reduce PCR artifacts, a maximum of three identical sequence reads was allowed. To perform mapping independently of DNA methylation status, sequence reads were in silico bisulfite-converted (C to T), and subsequently mapped to two different in silico converted mm9 genome sequences; one C to T converted genome and one G to A converted genome. Reads mapping to both genomes were discarded, which typically represented a very minor fraction of all reads. Mapping was done using the Burrows-Wheeler Aligner (BWA), with default settings allowing a single mismatch. The obtained mapping positions were used to align unconverted sequence reads with their corresponding unconverted genomic sequence and to subsequently determine the methylation status of each sequenced cytosine within a CpG context, both on the forward strand as well as on the reverse strand.
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| Submission date |
Oct 24, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Arjen Brinkman |
| E-mail(s) |
arjen.brinkman@gmail.com
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| Organization name |
Radboud University, Nijmegen Center for Molecular Life Sciences
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| Department |
Molecular Biology
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| Lab |
Stunnenberg
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| Street address |
NCMLS #274, Geert Grooteplein Zuid 30
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE28254 |
Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk |
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| Relations |
| SRA |
SRX105266 |
| BioSample |
SAMN00752489 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM821395_mES_Dnmt-TKO_H3K27m3_ChIP-BS.meth.bed.gz |
72.1 Mb |
(ftp)(http) |
BED |
| GSM821395_mES_Dnmt-TKO_H3K27m3_ChIP-BS.reads.bed.gz |
1.1 Gb |
(ftp)(http) |
BED |
| GSM821395_mES_Dnmt-TKO_H3K27m3_ChIP-BS.reads.wig.gz |
66.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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