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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 22, 2012 |
| Title |
E14 day4 MRE-seq |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells (E14)
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| Organism |
Mus musculus |
| Characteristics |
development stage: Day 4
cell type: mouse embryonic stem cells (E14)
treatment: activin
genetic background: 129P2
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| Treatment protocol |
Guided differentiation procedures were preformed as following: 2×105 cells were seeded on the Collagen IV coated 10cm dishes (BD, 08-774-33) in serum free medium ESF-B (Itochu Corporation) supplemented with 0.1%BSA, 50M 2-Me and 10 ng/ml Activin A. Mediums were changed every day. Genomic DNAs and total RNAs were collected in day0, 4, and 6.
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| Growth protocol |
E14(mESC) were plated onto gelatin-coated dishes in Dulbecco's modified Eagle medium (DMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum (FBS; GIBCO), 0.055 mM -mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF (Millipore ESG1107) in incubator set at 37 ºC in 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MRE-seq was performed as described (NATURE| Vol 466|8 July 2010). Four parallel digests were performed (HpaII, Hin6I and SsiI are from Fermentas; HpyCH4IV is from NEB), each with 1 ug of DNA. Five units of enzyme per microgram DNA were added and incubated at 37˚C for 3 hrs. A second dose of five units-enzyme was added and the DNA was incubated for an additional 3 hrs. Digested DNA was precipitated with sodium acetate and ethanol, and 500 ng of each digest were combined into one tube. Combined DNA was size-selected by electrophoresis on a 1% agarose gel. A 100-500 bp gel slice was excised and gel-purified using Qiagen Qiaquick columns, eluting in 30 ul of Qiagen EB buffer. Library construction was performed using the Illumina Genomic DNA Sample kit with paired-end adapters, following the manufacturer’s instructions with the following changes. For the end repair reaction, T4 DNA polymerase and T4 polynucleotide kinase were excluded and the klenow DNA polymerase was diluted 1:5 in water and 1 ul used per reaction. For paired-end oligo adapter ligation, adapters were diluted 1:10 in water and 1 ul used per reaction. After the second size selection, DNA was eluted in 36 ul EB buffer using Qiagen Qiaquick columns, and 12 ul used as template for PCR, using Illumina reagents and cycling conditions with 15 cycles. After cleanup with Qiagen MinElute columns, each library is examined by Quant-iT™ dsDNA HS Assay Kit (Qubit, Invitrogen, USA) and Agilent DNA Biaanalyzer (Agilent, USA). MeDIP-seq was performed as described (NATURE| Vol 466|8 July 2010). 3 ug DNA isolated as described above was sonicated to ,100–500 bp with a Bioruptor sonicator (Diagenode). Sonicated DNA was end-repaired, A-tailed, and ligated to paired-end adapters following the standard Illumina protocol. After agarose size-selection to remove unligated adapters, 1 ug of adaptor-ligated DNA was used for each immunoprecipitation using a mouse monoclonal anti-methylcytidine antibody (1 mg/ml, Eurogentec). For this, DNA was heat-denatured at 95 ˚C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 ul primary antibody per microgram of DNA overnight at 4 ˚C with rocking agitation in 500 ml immunoprecipitation buffer (10mMsodium phosphate buffer, pH 7.0, 140mMNaCl, 0.05% Triton X-100). To recover the immunoabsorbed DNA fragments, 2ul of rabbit anti-mouse IgG secondary antibody (2.5 mg/ml, Jackson Immunoresearch) and 100 ml Protein A/G beads (Pierce Biotechnology) were added and incubated for an additional 2 h at 4 ˚C with agitation. After immunoprecipitation a total of six to ten immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. A nonspecific mouse IgG immunoprecipitation (Jackson Immunoresearch) was performed in parallel to methyl DNA immunoprecipitation as a negative control. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/ml proteinase K for 2 h at 55 ˚C and then allowed to cool to room temperature. MeDIP and supernatant DNA were purified using Qiagen Qiaquic columns and eluted in 30 ul EB (Qiagen). Twelve cycles of PCR were performed on 10 ul of the immunoprecipitated DNA using the paired-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (192–592 bp) was performed by electrophoresis in 2% agarose. Libraries were quality controlled by Qubit and Agilent DNA Bioanalyzer analysis. An aliquot of each library was diluted in EB to 5 ng/ul and 1 ul used as template in four independent PCR reactions to confirm enrichment for methylated and de-enrichment for unmethylated sequences, compared to 5 ng of input (sonicated DNA). Two positive controls (SNRPN and MAGEA1 promoters) and two negative controls (a CpG-less sequence on chromosome 15 and GAPDH promoter) were amplified (Supplementary). Cycling was 95 ˚C for 30 s, 58 ˚C for 30 s, 72 ˚C for 30 s with 30 cycles. PCR products were visualized by 1.5% agarose gel electrophoresis. DNA product for 5-hmC_ChIP-seq is generated by a selctive chemical labeling method (Nat. Biotechnol. 2011, 29, 68-72). For ncRNA-seq, Total RNA was purified by Trizol Reagent (Invitrogen) and used as input to generate microRNA library by True-seq small RNA kit (Illumina). The RNA 3’ adapter is specifically modified to target microRNAs and other small RNAs that have a 3’ hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes. The bands of library products from 145-160bp were cut and amplified for sequencing. The libraries were then quantitated by qPCR.
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| Library strategy |
MRE-Seq |
| Library source |
genomic |
| Library selection |
Restriction Digest |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
The libraries for ncRNA and 5-hmC were sequenced on a HiSeq2000 using a TruSeq SBS sequencing kit version 2 and analyzed with pipeline version 1.8. The libraries of MeDIP-seq and MRE-seq were sequenced on Illumina GAII.
For small ncRNA sequencing data, The 3’ adapters were first trimmed from all 40bp short sequencing reads and short reads (<18bp) were removed after trimming. After that, reads were mapped into mouse mm9 genome using bowtie software with no mismatch in the first 18 seed sequences, and with no more than 5 alignments in the genome. Only the best alignments for each mappable reads were reported.
MRE-seq data were generated by index-sequencing(each index has 7bp), and total length of each sequenced read is 49bp. so the valid length of each read is 42bp. After trimming the indexes, the 42bp reads were mapped to mm9 genome by MAQ with an additional constraint that the 5′ end of a read must map to the CpG site within a MRE site
MeDIP-seq data were mapped to mm9 genome by MAQ
Sequence reads for 5hmC were mapped to mm9 using Bowtie. Only reads with one or fewer mismatches and uniquely mapped onto the genome were retained.
Genome_build: mm9
Supplementary_files_format_and_content: BED
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| Submission date |
Jun 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Pengfei Yu |
| E-mail(s) |
yupf05@gmail.com
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| Organization name |
University of California San Diego
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| Department |
Bioengineering
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| Lab |
Dr. Sheng Zhong
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| Street address |
9500 Gilman Drive MC 0419
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093-0419 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE38596 |
Genome-wide maps of cytosine methylation, cytosine hydroxylmethylation and small non coding RNAs in mouse ES cells and upon guided differentiation to mesoendoderm cells |
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| Relations |
| SRA |
SRX152131 |
| BioSample |
SAMN01041509 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM945902_E14_d4_MRE-seq.bed.gz |
524.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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