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| Status |
Public on Dec 15, 2012 |
| Title |
72h_SAG_H3K27ac |
| Sample type |
SRA |
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| Source name |
Neuralized embryoid bodies
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| Organism |
Mus musculus |
| Characteristics |
treatment: all trans retinoic acid (RA) and SAG for 3 days before harvested
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| Growth protocol |
mouse ESCs were cultured on MEFs in the presence of in LIF/15% FBS prior to differentiation. Cells were in vitro differentiated by suspension culture in DFNB or DNFK (DMEM:F12:Neurobasal=1:1:2 supplemented with 1x B27 or Knockout Serum Replacement) for two days. "t=0" samples were collected at this time (two days of suspension culture). Cells were induced with all-trans retinoic acid (RA, 500nM) with or without a small molecule agonist for Smo (SAG, 50-800nM). Samples were collected 3 days after RA or RA/SAG induction (t=72h RA and t=72h RA/SAG).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin precipitation protocol was previously described (Odom et al. 2006; Vokes et al. 2007). A previously characterized Gli1FLAG cell line that constitutively expresses Gli1FLAG cDNA under Rosa26 transcriptional regulation was used for Gli1FLAG ChIP (Vokes et al. 2007). EBs were dissociated in trypsin then crosslinked in 1% formaldehyde at room-temperature for 30 minutes. Cells were lysed in lysis buffer (140mM NaCl, 0.5% NP40, 0.25% Triton-X 100, 10% glycerol, 1mM EDTA, 50mM HEPES-KOH, pH7.5) or (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) at 4˚C with agitation for 10 minutes then washed with Buffer 2 (200mM NaCl, 1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0) for 10 minutes at room-temperature. Chromatin was fragmented by sonication (Branson digital sonifier with microtip) in Buffer 3 (1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0). Lysate was cleared by centrifugation and supplemented with NaCl, NaDeoxycholate, and TritonX 100 to the final concentrations of 100mM, 0.1%, and 1%, respectively. Magnetic beads (Dynal/Invitrogen, sheep anti-mouse IgG, anti-rabbit IgG, and Protein G for mouse, rabbit, and goat antibodies, respectively) were blocked with a blocking buffer (5mg/ml BSA in PBS) then incubated with an antibody in the blocking buffer overnight. Unbound antibody was washed away by extensive wash with the blocking buffer. Beads/antibody complex was incubated with the lysate overnight at 4°C. Beads were washed 5 times with RIPA buffer (1% NP40, 0.7% NaDeoxycholate, 0.5M LiCl, 1mM EDTA, 50mM HEPES-KOH, pH7.5). DNA was eluted with elution buffer (1% SDS, 1mM EDTA, 50mM Tris-Cl, pH8.0) at 65˚C for 15 min, and the eluate was incubated further overnight at 65˚C to reverse crosslinking. DNA was purified by phenol/chloroform extraction and ethanol precipitation or by PCR purification kit (Qiagen). All buffers except RIPA buffer was supplemented with protease inhibitor cocktail mix (Roche).
Libraries were prepared according to Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Embryoid bodies were treated with all trans retinoic acid (RA) and SAG for 3 days before harvested for ChIP analysis (see Growth Protocol section)
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| Data processing |
Base calling was performed using Illumina pipeline
The firsty 25 bp of sequence reads were mapped to unmasked mouse genome using either seqmap or eland allowing up to two mismatches
Peak calling was performed using a modified version of CisGenome peak caller (FDR0.01)
Independent ChIP sampes for YFP (mock FLAP-IP) and Foxa2 were combined prior to peak calling
Histone tag counts were generated using Homer makeTagDirectory function
Genome_build: mm9
Supplementary_files_format_and_content: Processed files include bed files for peak predictions and sequence tag counts for histone data generated by Homer makeTagDirectory function
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| Submission date |
Nov 07, 2012 |
| Last update date |
May 16, 2019 |
| Contact name |
Kevin Peterson |
| E-mail(s) |
kevin.peterson@med.usc.edu
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| Phone |
323-442-8077
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| Organization name |
Eli and Edythe Broad-CIRM Center for Regenerative Medicine, University of Southern California Keck School of Medicine
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| Department |
Department of Stem Cell Biology and Regenerative Medicine
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| Lab |
Andrew McMahon
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| Street address |
1425 San Pablo St.
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE42132 |
ChIP-seq analysis of Gli1 and Sox2 input to neural progenitor program and integration with actve histone marks |
| GSE42594 |
cis-Regulation of Shh-directed neural pattering |
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| Relations |
| SRA |
SRX203365 |
| BioSample |
SAMN01804586 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1033103_72h_SAG_H3K27ac.all.tags.tsv.gz |
164.7 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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