|
| Status |
Public on Oct 15, 2014 |
| Title |
Eomesa_ChIP1 |
| Sample type |
SRA |
| |
|
| Source name |
high-sphere stage embryos
|
| Organism |
Danio rerio |
| Characteristics |
strain: AB
developmental stage: high-sphere
chip antibody: Eomesa
|
| Treatment protocol |
Embryos fixed in 1.85% formaldehyde for 15 mins prior to chromatin extraction
|
| Growth protocol |
Embryos grown in E3 embryo medium at 28.5 degrees celcius
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sequential cytoplasmic and nuclear lysis, followed by sonication and centrifugal clarification from was used to obtain chromatin of appropriate fragment size. Desired protein-DNA complexes were isolated using antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Sequence reads were aligned to the Zv9 mouse genome release using Bowtie with the following options: -n2 –e120 –l 28 -m2 -k2 -best
Peaks were called in ChIP samples over input using MACS with the following options: −−mfold8 –pvalue 1e-4
Peak coordinates common to both ChIP replicates were identified for further analysis using custom Perl scripts
Genome_build: Zv9
Supplementary_files_format_and_content: peak wig files were generated using MACS. Peak bed files were generated using MACS followed by custom Perl scripts.
|
| |
|
| Submission date |
Oct 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew Christopher Nelson |
| E-mail(s) |
a.nelson.1@warwick.ac.uk
|
| Organization name |
University of Warwick
|
| Department |
School of Life Sciences
|
| Street address |
Gibbet Hill Campus
|
| City |
Coventry |
| ZIP/Postal code |
CV4 7AL |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9319 |
| Series (2) |
| GSE51891 |
Genome-wide profiling of Smad2 and Eomesa binding in zebrafish blastulas |
| GSE51894 |
Smad2 and Eomesa |
|
| Relations |
| BioSample |
SAMN02389005 |
| SRA |
SRX370997 |
