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| Status |
Public on Jul 04, 2013 |
| Title |
ChIP-seq analysis of induced V5-tagged Lhx3 (iLhx3-V5) in iNIL3 induced motor neurons (Day 4) |
| Sample type |
SRA |
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| Source name |
induced motor neurons (Day4 iNIL differentiation)
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| Organism |
Mus musculus |
| Characteristics |
cell line: Ainv15 (ATCC SCRC-1029) with Dox-inducible Ngn2.Isl1.Lhx3-V5 construct
cell treatment: +Dox 48hrs
cell stage: induced motor neurons (Day4 iNIL differentiation)
chip antibody: anti-V5 (Abcam, ab15828, Lot: 103968)
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| Treatment protocol |
Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 uM all-trans-Retinoic acid (RA, Sigma) and 0.5 uM agonist of hedgehog signaling (SAG, Calbiochem). Doxycycline (Sigma) was added to the culture medium at 3ug/ml 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 5x105 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 2 and Day 5 of differentiation. Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 uM all-trans-Retinoic acid (RA, Sigma) and 0.5 uM agonist of hedgehog signaling (SAG, Calbiochem). For ChIP experiments, the same conditions were used but scaled to seed 1x107 cells on Day 0. Doxycycline (Sigma) was added to the culture medium at 3ug/ml when required.
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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 x106 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-Isl1 monoclonal antibodies are a generous gift from Dr. T. Jessell (Columbia University). 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 V5
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| Data processing |
Alignment: Sequence reads were aligned to the mouse genome (version mm9) using Bowtie (Langmead, et al. Genome Biology 2009) version 0.12.5 with options "-q --best --strata -m 1 -p 4 --chunkmbs 1024". Only uniquely mapping reads were analyzed further. All alignment data is provided here in BAM format.
Peaks: Binding events were detected using GPS (Guo, et al. Bioinformatics 2010) version 1.0. 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. Peaks are provided here in two formats, BED and GPS raw output. The BED format shows the GPS-predicted binding position, and the Q-value calculated by GPS is provided in the score field. The GPS raw output has the following fields (tab-separated): 1) Binding event coordinate, 2) IP read count assigned to event, 3) Control read count assigned to event, 4)Fold enrichment (IP/Control), 5) Q-value (multiple hypothesis corrected), 6) P-value, 7) Shape deviation from the empirical read distribution (log10(KL)), 8) Shape deviation between IP vs Control (log10(KL)).
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 Isl1-2_iMN_Day4 eexperiment due to lower libnrary complexity than sequencing dpeth. WIG files were converted to bigWig using wigToBigWig (UCSC)
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| Submission date |
Aug 19, 2011 |
| 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) |
| GSE31456 |
Transcriptional mechanisms controlling direct motor neuron programming |
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| Relations |
| SRA |
SRX092470 |
| BioSample |
SAMN00710410 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM782847_iMN.ES-MN_iNgn2.Isl1.Lhx3_Day4+Dox-RA-Hh.ChIP-seq_iLhx3-V5_rep1.bam |
1.2 Gb |
(ftp)(http) |
BAM |
| GSM782847_iMN.ES-MN_iNgn2.Isl1.Lhx3_Day4+Dox-RA-Hh.ChIP-seq_iLhx3-V5_rep1.bw |
275.0 Mb |
(ftp)(http) |
BW |
| GSM782847_iMN.ES-MN_iNgn2.Isl1.Lhx3_Day4+Dox-RA-Hh.ChIP-seq_iLhx3-V5_rep1.peaks.bed.gz |
328.3 Kb |
(ftp)(http) |
BED |
| GSM782847_iMN.ES-MN_iNgn2.Isl1.Lhx3_Day4+Dox-RA-Hh.ChIP-seq_iLhx3-V5_rep1.peaks.gps.txt.gz |
569.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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