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| Status |
Public on Jul 04, 2013 |
| Title |
Pol2 in progenitor motor neurons [ChIP-seq] |
| Sample type |
SRA |
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| Source name |
progenitor motor neurons
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| Organism |
Mus musculus |
| Characteristics |
cell line: HBG3
cell-treatment: +RA +Hh
cell-stage: progenitor motor neurons (Day 4)
chip-antibody: anti-Pol2 antibody 8WG16 (Covance MMS-126R)
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| Treatment protocol |
Ectopic exposure to retinoic acid and hedgehog agonist at day 2 during the differentiation protocol.
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| Growth protocol |
ES cells were differentiated as previously described (Wichterle, et al. Cell 2002). Briefly, ES cells were trypsinized 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). Medium was exchanged on Day 1 and Day 2 of differentiation. 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). 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 |
ChIP protocols were adapted from http://jura.wi.mit.edu/young_public/hESregulation/ChIP.html. Descriptions of these protocol modifications have been previously published (Guenther, et al., Genes Dev 2008). Briefly, approximately 6x10^7 cells taken from each developmental time point were cross-linked using formaldehyde and snap-frozen in liquid nitrogen. Cells were thawed on ice, resuspended in 5ml lysis buffer 1 (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and mixed on a rotating platform at 4°C for 5 minutes. Samples were spun down for 3 minutes at 3000rpm, resuspended in 5ml lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), and mixed on a rotating platform for 5 minutes at room temperature. Samples were spun down once more, resuspended in lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp. Triton X-100 was added to the lysate after sonication to final concentrations of 1% and the lysate spun down to pellet cell debris. The resulting whole-cell extract supernatant was incubated on a rotating mixer overnight at 4°C with 100 µL of Dynal Protein G magnetic beads that had been preincubated for 24 hours with 10 µg of the appropriate antibody in a PBS/BSA solution. After approximately 16 hours of bead-lysate incubation, beads were collected with a Dynal magnet. ChIP samples probing for transcription factor binding were washed with the following regimen, mixing on a rotating mixer at 4°C for 5 minutes per buffer: low-salt buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high-salt buffer (20 mM Tris at pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris at pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% deoxycholate, 1% NP-40), and TE containing 50 mM NaCl. ChIP samples probing for histone and chromatin marks were washed 4 times with RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) and then once with TE containing 50 mM NaCl, again mixing on a rotating mixer at 4°C for 5 minutes per buffer. After the final bead wash, samples were spun down to collect and discard excess wash solution, and bound antibody-protein-DNA fragment complexes were eluted from the beads by incubation in elution buffer at 65°C with occasional vortexing. Cross-links were reversed by overnight incubation at 65°C. Samples were digested with RNase A and Proteinase K to remove proteins and contaminating nucleic acids, and the DNA fragments precipitated with cold EtOH. Purified DNA fragments were processed according to a modified version of the Illumina/Solexa sequencing protocol (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 Pol2
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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. WIG files were converted to bigWig using wigToBigWig (UCSC).
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 (1) |
| GSE39434 |
Chromatin landscape of progenitor motor neurons [ChIP-seq] |
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| Relations |
| SRA |
SRX160502 |
| BioSample |
SAMN01090693 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM968717_pMN_Day4+RA+Hh.ChIP-seq_Pol2_rep1.bw |
157.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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