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| Status |
Public on Aug 24, 2015 |
| Title |
Gli3FLAG_NPC_ChIP-seq |
| Sample type |
SRA |
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| Source name |
Neural progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6 X 129/sv
genotype/variation: 3xFLAG-Avi tag insertion at Gli3 initiation codon in V6.5
cell type: ES-derived neural progenitors (dorsal)
chip antibody: anti-FLAG M2 clone
chip antibody vendor: Sigma
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| Growth protocol |
Ventral neural progenitors were routinely generated from mouse ESCs by two days of suspension culture in DFNB (25% DMEM, 25% F12, 50% Neurobasal supplemented with 1xB27 [Life Technologies]) or DFNK (10% Knockout Serum Replacement [Life Technologies] in place of B27 in DFNB) followed by additional three days in DFNB with 500nM all-trans retinoic acid and 160-800nM Smoothened agonist (SAG, Calbiochem/EMD Millipore). To obtain dorsal characteristics in Gli3 profiling, SAG was omitted from the induction formula.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells 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).
Sequencing library was constructed using ChIP-Seq DNA Sample Prep Kit (Illumina IP-102-1001) according to the manufacturer's instruction. Briefly, DNA was blunted and phosphorylated with T4 and Klenow DNA polymerases and T4 PNK followed by adapter ligation with T4 DNA ligase. A range of 275-700bp was selected by gel purification and amplified with PCR primers 1.1 and 2.1 for 18 cycles.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample 4
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| Data processing |
Basecalls performed using Illumina pipeline
The first 25bp of ChIP-seq reads were aligned to the unmasked mm9 genome using Eland allowing up to two mismatches (Samples 1-3, 507) or aligned against mm9 with Bowtie2 (version 2.2.4) to obtain SAM (Samples 4,8-13).
Unique reads were used to identify peaks using CisGenome peak caller in the 2-sample conditional binomial mode against matched input DNA data at FDR=0.01 cutoff
Heatmap comparison of rep1 and rep2 was performed by HOMER to generate _heatmap.txt output with the default settings.
Genome_build: mm9
Supplementary_files_format_and_content: bed files contain binding regions called in this analysis; heatmap.txt output file for heatmap comparision of rep1 and rep2
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| Submission date |
Sep 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuichi Nishi |
| E-mail(s) |
yuichi.nishi@gmail.com
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| Phone |
323-442-8077
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| Organization name |
University of Southern California
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| Department |
Eli and Edythe Broad-CIRM Center for 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) |
| GSE61673 |
Nkx2.2, Nkx6.1, Olig2, and Gli3 genomic binding regions in neural progenitors [ChIP-Seq] |
| GSE65462 |
Nkx2.2, Nkx6.1, Olig2, and Gli3 genomic binding regions and overexpression in neural progenitors |
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| Relations |
| BioSample |
SAMN03076121 |
| SRA |
SRX708732 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1511110_Gli3.bed.gz |
17.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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