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| Status |
Public on Nov 07, 2014 |
| Title |
st10.5_Smad23_ChIP_rep1 |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Xenopus tropicalis |
| Characteristics |
strain: Nigerian
developmental stage: mid-gastrula stage (st10.5)
chip antibody: mouse anti-Smad2/3 antibody
chip antibody vendor: BD
chip antibody cat. #: #610842
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| Treatment protocol |
Two thousand embryos were cultured until early gastrula (stage 10.5). Embryos were crosslinked in batches of 200 embryos with 1ml 1% formaldehyde in 1/9XMMR at room temperature for 45 minutes with gentle rocking. Crosslinking reactions were neutralized by removal of the formaldehyde solution and incubation in 1ml 0.125M glycine for 5 minutes at room temperature. Processed embryos were washed twice with ice-cold RIPA buffer (50 mM Tris-HCl pH7.4, 150mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate, 1% NP40, 0.1% SDS, 0.5 mM DTT, and Roche cOmplete protease inhibitor cocktail). The samples can be flash-frozen in liquid nitrogen and stored at -80°C
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| Growth protocol |
Embryos were cultured in 1/9X MMR at 25°C until the indicated stage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The fixed embryos were homogenized with an Eppendorf tube pestle and microfuged at 14,000rpm, 4°C for 15 minutes. The pellets, which contain chromatin, were re-suspended in 750ul RIPA buffer and sonicated on ice using a Branson Digital Sonifier 450 (18% power output, 20 sec per pulse, 10 pulses) resulting in DNA fragmentation with a maximum between 200-500bp long. The resulting samples were microfuged at 4°C and 14,000 rpm for 20 minutes to remove insoluble cellular debris. Supernatant containing fragmented chromatin was “pre-cleared” by incubating with 20μl Protein A-coated Dynabeads (Invitrogen) for 2 hour at 4°C. 50 embryo equivalent (eeq) volume of the chromatin supernatant was frozen for use as “input control”. Antibodies were pre-bound to 20μl of Protein A Dynabeads (per batch of 200 embryos) by incubating at 4°C for 30 min. Sonicated chromatin fragments were then added to antibody-bound Dynabeads and incubated overnight at 4°C on an end-over-end rotator. The next day, supernatants were discarded, the recovered beads were washed once with ice-cold ChIP wash solution I (50mM HEPES-KOH pH7.5, 2mM EDTA, 150mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1mM DTT, and 0.4mM PMSF), ChIP wash solution II (50mM HEPES-KOH pH7.5, 2mM EDTA, 500mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1mM DTT, and 0.4mM PMSF), and ChIP wash solution III (0.25 M LiCl, 1 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM DTT, and 0.4 mM PMSF), and finally ice-cold TE (10mM Tris, 1mM EDTA, 1 mM DTT, and 0.4 mM PMSF). The DNA was then eluted with TE buffer containing 1% SDS, and reverse-crosslinked at 65°C for 6 hours to overnight. Frozen sonicated input control chromatin frozen was diluted with elution buffer (3-fold) and also incubated at 65°C. On the following day the samples were treated with RNAse A (0.2μg/μl) for 2hr at 37°C, followed by Proteinase K (0.2μg/μl) for 2hr at 55°C, followed by phenol/chloroform extraction and ethanol precipitation. DNA pellets were resuspended in Qiagen EB solution. For ChIP-qPCR analyses on enriched chromatin fragments, immunoprecipitated DNA was amplified using specific primers to detect enrichment in the denoted genomic regions. Results were normalized against input DNA.
Libraries were prepared using Bioo Scientific NEXTflex™ ChIP-seq Kit (Cat# 5143-01)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
processed data file: st_10.5_Smad2_peaks_MACS_SISSRs_overlap.txt
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| Data processing |
Raw ChIP-seq reads from the replicates were pooled and mapped to an indexed Xenopus tropicalis genome using Bowtie (v. 0.12.9) (Langmead et al. 2009), only uniquely mapped reads were retained, i.e. parameters: -m 1
MACS (v. 2.0.10) (Zhang et al. 2008) and SISSRs (v. 1.4) (Jothi et al. 2008) were used for peak calling. Effective genome size is 1.16e+09. MACS was run with default setting, Sissrs was run with parameter -a
Peaks were assigned to the nearest genes, using BEDTools (v 2.16.2) (Quinlan and Hall 2010), based on the following criteria: peak summits need to lie within 10 kb upstream of the 5’ end, within the gene body (5’ UTRs, exons, introns, and 3’UTRs), or 10 kb downstream of the 3’ end of the gene.
Genome_build: Xenopus tropicalis genome build v 7.1
Supplementary_files_format_and_content: bed format of peaks only called by both MACS and Sissrs
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| Submission date |
Dec 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
William Chiu |
| E-mail(s) |
wtychiu@uci.edu
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| Organization name |
University of California, Irvine
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| Department |
Developmental and Cell Biology
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| Lab |
Cho
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| Street address |
Room 4410 Natural Sciences II
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| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
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| Platform ID |
GPL15472 |
| Series (2) |
| GSE53652 |
Genome-wide view of TGFb/Foxh1 regulation of the early mesendoderm program [ChIP-seq] |
| GSE53654 |
Genome-wide view of TGFb/Foxh1 regulation of the early mesendoderm program |
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| Relations |
| Reanalyzed by |
GSE85273 |
| BioSample |
SAMN02486392 |
| SRA |
SRX399449 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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