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| Status |
Public on Jul 04, 2013 |
| Title |
H3K27me3 in RA/Hh-derived motor neurons (Day5) exposed to Dox(iCdx2), RA, Hh and FGF [ChIP-Seq] |
| Sample type |
SRA |
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| Source name |
motor neurons (Day 5)
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| Organism |
Mus musculus |
| Characteristics |
cell-line: Ainv15 (ATCC SCRC-1029) with Dox-inducible Cdx2-V5 construct
cell-treatment: +RA +Hh +Dox +FGF
cell-stage: motor neurons (Day 5)
chip-antibody: anti-H3K27me3 (Abcam, ab6002)
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| Treatment protocol |
Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 µM all-trans-Retinoic acid (RA, Sigma) and 0.5 µM agonist of hedgehog signaling (SAG, Calbiochem). 2ug/ml Doxycycline (Sigma) and 100 ng/ml bFGF (PeproTech) were added to the culture medium on Day 3 when required.
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| Growth protocol |
ES cells were cultured over a layer of Mitomycin-C treated fibroblast resistant to Neomycin (Fisher) in EmbryoMax D-MEM (Fisher) supplemented with 10% ES-FBS (Invitrogen), L-Glutamine (Gibco), 0.1 mM beta-mercaptoethanol and 100 U/ml LIF. Motor neuron differentiation of ES cells was performed as previously described. Briefly, ES cells were trypsinized (Invitrogen) and seeded at 5x10^5 cells/ml in ANDFK medium (Advanced DMEM/F12:Neurobasal (1:1) Medium, 10% Knockout-SR, Pen/Strep, 2 mM L-Glutamine, and 0.1 mM 2-mercaptoethanol) to initiate formation of embryoid bodies (Day 0). Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 µM all-trans-Retinoic acid (RA, Sigma) and 0.5 µM agonist of hedgehog signaling (SAG, Calbiochem). On Day 3, cultures were treated with Doxycycline (Dox) (2 µg/ml) for 6 to 8 hours. Cultures were collected, washed with PBS, and treated with 100 nM RA, 100 ng/ml bFGF (PeproTech), and 100 nM Hh agonist and medium was changed on Day 5. For ChIP experiments, the same conditions were used but scaled to seed 1x10^7 cells on Day 0.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 minutes at room temperature. Pellets containing ~40 x10^6 cells were flash frozen and stored at -80 oC. Cells were thawed on ice, resuspended in 5ml of Lysis Buffer A and incubated for 10 minutes at 4 oC in a rotating platform. Samples were spun down for 5 minutes at 1,350g, resuspended in 5ml Lysis Buffer B and incubated for 10 minutes at 4 oC in a rotating platform. Samples were spun down for 5 minutes at 1,350g, resuspended in 3ml of Sonication Buffer (SB). Nuclear extracts were sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp. Sonicated chromatin was incubated for 16 hours at 4C with Protein-G beads (Invitrogen) conjugated with either rabbit anti-V5 (Abcam) or anti-H3K27me3 (Abcam). After incubation and with the aid of a magnetic device, beads were washed once with SB+500nM NaCl, once with LiCl Wash Buffer and 1ml of TE. Then, beads were centrifugated at 950g for 3 min and remove residual TE with a pipette. 210ul of Elution Buffer was added to the beads followed by incubation at 65 oC for 45 minutes with a brief pulse of vortex every 10 minutes. 200ul of supernatant was removed after a 1 minute centrifugation at 16,000g. The crosslink was reversed by 16 hours incubation at 65 oC. RNA was digested by the addition of 200ul of TE and RNAseA (Sigma) at a final concentration of 0.2mg/ml and incubated for 2 hours at 37C. Protein was digested by the addition of Proteinase K (0.2 mg/ml final, Invitrogen) supplemented with CaCl2 followed by a 30 minutes incubation at 55 oC. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and then recovered with an ethanol precipitation with glycogens as carrier. The pellets were suspended in 70ul of water. Purified DNA fragments were processed according to the Illumina/Solexa library protocol and sequenced using a Genome Analyzer II (Illumina, http://www.illumina.com/pages.ilmn?ID=252).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Chromatin IP against H3K27me3
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| Data processing |
Basecalling: Performed with CASAVA v1.7
BIGWIG: WIG files were generated using custom software to summarize read counts overlapping genomic regions. All reads were artificially extended out to 200bp in length. A bin size of 20bp was used for calculating overlapping read counts. Read counts per starting base pair were limited to 1 in the H3K27me3 experiments due to lower library complexity than sequencing depth. WIG files were converted to bigWig using wigToBigWig (UCSC).
Peaks: TF binding events were detected using GPS (Guo, et al. Bioinformatics 2010) version 2.0. In GPS, the scaling ratio between IP and control channels was estimated using the median ratio of all 10Kbp windows along the genome. The GPS binding model was initialized to the default and iteratively updated over up to 3 training rounds. In this study, we require that reported peaks contain a ChIP-seq enrichment level that is significantly greater than 1.5 times the control level with p-value <0.01 as tested using the Binomial distribution. Signal-to-noise ratios are estimated by comparing the ChIP-seq read count occurring at any peak found for a given transcription factor in any condition to the count of remaining reads in that experiment.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files are generated from aligned reads as described in the data processing steps.
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| Submission date |
Jul 17, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
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| Lab |
Shaun Mahony
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| Street address |
404 South Frear Bldg
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (2) |
| GSE39433 |
Induced Cdx2 binding in progenitor motor neurons and its effect on H3K27me3 chromatin domains [ChIP-Seq] |
| GSE39453 |
Induced Cdx2 binding in progenitor motor neurons and its effect on H3K27me3 chromatin domains |
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| Relations |
| SRA |
SRX160497 |
| BioSample |
SAMN01090688 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM968712_iCdx2-V5_Day4+Dox+RA+Hh+FGF.ChIP-seq_H3K27me3_rep1.bw |
178.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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