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| Status |
Public on Mar 30, 2017 |
| Title |
stage_8_Input_DNA |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Xenopus tropicalis |
| Characteristics |
strain: Nigerian
Stage: st. 8
chip antibody: None
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| Treatment protocol |
Embryos were fixed in 1% formaldehyde in 1/9XMMR at room temperature for 45 minutes with gentle rocking. Crosslinking reactions were neutralized by the removal of the formaldehyde solution and incubation with 1ml 0.125M glycine solution for 10 minutes on ice. Embryos were then washed twice with 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). Embryos were 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° until the indicated stage.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The fixed embryos were homogenized in RIPA buffer and incubated on ice for 10 minutes. Samples were then microfuged at 14,000rpm for 15 minutes at 4°C. Pellets were resuspended in RIPA buffer and sonicated on ice to an average size of 200-500bp. The resulting samples were microfuged at 14,000 rpm for 20 minutes at 4°C to remove insoluble cellular debris. The sheared chromatin was “pre-cleared” by incubating with Protein A-coated Dynabeads (Invitrogen) for 2 hour at 4°C with rotation. Antibodies were pre-bound to blocked Protein A Dynabeads by incubating at 4°C for 30 min. A sample of sheared chromatin was frozen for use as input control. Pre-cleared chromatin was added to antibody-bound Dynabeads, and incubated overnight at 4°C on an end-over-end rotator. The next day, the beads were washed, and DNA was eluted off the beads with TE buffer containing 1% SDS, and reverse-crosslinked at 65°C overnight. Sonicated input control was diluted 3-fold with elution buffer and also incubated at 65°C. All samples were treated with RNAse A, Proteinase K, phenol/chloroform extracted, and ethanol precipitated overnight. DNA pellets were resuspended in Qiagen EB solution.
10-30ng of DNA was used for library construction using the Bioo Scientific NEXTflex™ ChIP-seq Kit (Bioo Scientific #5143-01). Stage 8 Tle library was constructed using the TruSeq ChIP Sample Prep kit (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
ChIP-seq reads were aligned to the Xenopus tropicalis genome v7.1 using Bowtie v1.0.0 (Langmead et al., 2009). All multimapping reads were discarded.
Foxh1 and Smad2/3 ChIP-seq peaks were called using the Irreproducibility Discovery Rate (IDR) framework (Li et al., 2011) and MACS2 v2.0.10 (Zhang et al., 2008). All other samples were peak-called using MACS2 v2.0.10. MACS2 'callpeak' command was used against the stage-matched inut control.
Genome_build: Xenopus tropicalis v7.1
Supplementary_files_format_and_content: Bedgraph files were created using Samtools (Li et al., 2009) and Homer v4.7 (Heinz et al., 2010). Duplicate reads were removed from sorted BAM files using the ‘rmdup’ command in Samtools and biological replicates were concatenated. Bedgraph files were created using HOMER after first making a tag directory. Default conditions were applied for the generation of the bedgraph files (http://homer.salk.edu/homer/ngs/ucsc.html).
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| Submission date |
Aug 06, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Rebekah M Charney |
| E-mail(s) |
rcharney@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 |
Ken Cho
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| Street address |
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 |
GPL21875 |
| Series (1) |
| GSE85273 |
Foxh1 marks the embryonic genome prior to the activation of the mesendoderm gene regulatory program |
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| Relations |
| BioSample |
SAMN05525294 |
| SRA |
SRX2007801 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2263598_st_8_Input.ucsc.bedGraph.gz |
46.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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