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| Status |
Public on Dec 01, 2016 |
| Title |
Gmnn-ESC-Rep1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
Gmnn ChIP in ESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells (ESCs)
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| Treatment protocol |
no treatment
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| Growth protocol |
The A2lox mouse ES cell line (Iacovino et al., Stem Cells 2011, 29(10): 1580-88) and a clonal derivative engineered for Doxycycline-dependent Gmnn shRNA.expression (Yellajoshyula et al., PNAS 2011, 108(8): 3294-99) were routinely propagated on feeder cells (mouse embryonic fibroblast cells, Chemicon) in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Hyclone), 1 mM sodium pyruvate, 1 mM Non-Essential amino acids, 1mM L-Glutamine (Invitrogen), 10−4 M 2-mercaptoethanol (Sigma) and 103 units per ml of leukemia inhibitory factor (ESGRO, Chemicon).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Quantitative ChIP was done with modifications to a standard protocol (Upstate/Millipore) as follows. For each ChIP reaction, sheared chromatin (sonicated to 200-500 bp) from 2-5x10^6 mES cells fixed in 1% Formaldehyde (Sigma) in DMEM+5% FBS was incubated with 5µg antibody using Dynabeads (Invitrogen) as per the manufacturers instructions. Antibodies used for ChIP were Gmnn (N-18;Santa Cruz sc-8449) and H3K9ac (Abcam; Ab10812). After washing, elution and cross-link reversal, DNA from each ChIP sample and the corresponding input sample was purified. ChIP enrichement for positive control amplicons was analyzed further using qPCR as follows: each ChIP sample and a range of dilutions of the corresponding input sample (0.01%-5% input) were quantitatively analyzed with gene specific primers using the 7500 FAST Real-time PCR Detection System (ABI) and SYBR Advantage qPCR Premix (Clontech).
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| Label |
Cy3
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| Label protocol |
Cy3 nad Cy5 labeling of experimental (IP) and control (input) samples was performed by Mogene, LC, using the NimbleGen DualColor DNA Labeling Kit
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| Channel 2 |
| Source name |
Input for ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells (ESCs)
|
| Treatment protocol |
no treatment
|
| Growth protocol |
The A2lox mouse ES cell line (Iacovino et al., Stem Cells 2011, 29(10): 1580-88) and a clonal derivative engineered for Doxycycline-dependent Gmnn shRNA.expression (Yellajoshyula et al., PNAS 2011, 108(8): 3294-99) were routinely propagated on feeder cells (mouse embryonic fibroblast cells, Chemicon) in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Hyclone), 1 mM sodium pyruvate, 1 mM Non-Essential amino acids, 1mM L-Glutamine (Invitrogen), 10−4 M 2-mercaptoethanol (Sigma) and 103 units per ml of leukemia inhibitory factor (ESGRO, Chemicon).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Quantitative ChIP was done with modifications to a standard protocol (Upstate/Millipore) as follows. For each ChIP reaction, sheared chromatin (sonicated to 200-500 bp) from 2-5x10^6 mES cells fixed in 1% Formaldehyde (Sigma) in DMEM+5% FBS was incubated with 5µg antibody using Dynabeads (Invitrogen) as per the manufacturers instructions. Antibodies used for ChIP were Gmnn (N-18;Santa Cruz sc-8449) and H3K9ac (Abcam; Ab10812). After washing, elution and cross-link reversal, DNA from each ChIP sample and the corresponding input sample was purified. ChIP enrichement for positive control amplicons was analyzed further using qPCR as follows: each ChIP sample and a range of dilutions of the corresponding input sample (0.01%-5% input) were quantitatively analyzed with gene specific primers using the 7500 FAST Real-time PCR Detection System (ABI) and SYBR Advantage qPCR Premix (Clontech).
|
| Label |
Cy5
|
| Label protocol |
Cy3 nad Cy5 labeling of experimental (IP) and control (input) samples was performed by Mogene, LC, using the NimbleGen DualColor DNA Labeling Kit
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| Hybridization protocol |
ChIP samples and corresponding input chromatin samples were hybridized to Nimblegen HD2 2.1M feature tiled arrays after linear amplification (using the Sigma WGA kit (#WGA2)) and labelling as described above. MOgene performed ChIP-chip hybridizations using a NimbleGen Hybridization System and Kit, as recommended by the manufacturer.
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| Scan protocol |
MOgene performed scanning, using the NimbleGen MS 200 Microarray Scanner for two color microarrays
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| Data processing |
Data analysis was performed by MOgene, using Nimblegen platform software (Nimblescan/Signal Map). The Nimblescan FindPeak software generated scaled log2-ratio data (ratio of IP vs input for individual probes) peak files containing regions of significant factor binding/histone modification.
Peaks with fdr>0.2 were filtered from the list by Nimblescan. For Geminin ChIP, we set a cutoff of fdr<0.05 for each peak list and truncated peaks that did not meet this criteria. Peak intersection analysis for the three independent biological replicates for each sample type above was performed using Bedtools, peaks present in at least two of the three replicates were defined in ENSEMBL Build 67 (mm9), using Liftover, and the nearest transcription start site was defined with Peak Annotator, defining reproducible regions of peak overlap between the three experimental replicates. To define Geminin-dependent H3K9ac peaks, we performed the same analysis as above to define H3K9ac peaks found in 2 of 3 independent experiments, but retained the default cut-off of fdr>0.2 (which also indicates high quality peaks), to maintain all significantly enriched regions for comparisons with Gem binding. This analysis was done for three independent experimental samples in 2.5 day neuroectoderm both under wild type conditions (no Dox) and under conditions of Geminin knockdown (+Dox). Peaks remaining under conditions of Geminin knockdown were then subtracted from the no Dox sample, so define the “no Dox-specific” peaks that were lost upon Geminin knockdown.
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| Submission date |
May 15, 2016 |
| Last update date |
Dec 01, 2016 |
| Contact name |
Kristen L Kroll |
| E-mail(s) |
[email protected]
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| Organization name |
Washington University School of Medicine
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| Department |
Developmental Biology
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| Lab |
Kristen Kroll
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| Street address |
320 McDonnell Sciences/660 S. Euclid Ave.
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL16158 |
| Series (2) |
| GSE81450 |
Geminin promoter occupancy and Geminin-dependent histone acetylation during neuroectodermal cell specification |
| GSE81595 |
Genome-wide association of Geminin and Zic1 during neuroectodermal cell specification |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2152657_463256_Slot2_ES_GemChIP_WU_Kroll_2011-02-18_ratio_peaks.gff.gz |
822.4 Kb |
(ftp)(http) |
GFF |
| GSM2152657_463256_Slot2_WU_Kroll_2011-02-18_532_GmnnES_ChIP_Rep1.pair.gz |
42.6 Mb |
(ftp)(http) |
PAIR |
| GSM2152657_463256_Slot2_WU_Kroll_2011-02-18_635_GmnnES_ChIP_Rep1.pair.gz |
42.4 Mb |
(ftp)(http) |
PAIR |
| GSM2152657_ESGemChIP_R1_fdrpt05_mm9_USE.txt.gz |
70.4 Kb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
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