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| Status |
Public on Jul 02, 2015 |
| Title |
CM_CtermGFP_hr02 |
| Sample type |
SRA |
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| Source name |
GtaC-GFP 1h developing ; replicate 2
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| Organism |
Dictyostelium discoideum |
| Characteristics |
strain: AX2
genotype: GFP-GtaC/gtaC-
replicate: 2
developmental time: 1
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| Treatment protocol |
ChIP-seq: development on nitrocellulose filters placed on a buffer-soaked filter paper pad, cells were washed twice with potassium phosphate buffer (KK2) and deposited on nitrocellulose filter 3.3x106 cells/cm3
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| Growth protocol |
Wild-type and the different gtaC alleles strains were cultured axenically in HL5 supplemented with antibiotics.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: After cellular fixation, we isolated nuclei and prepared nuclear extract by sonication. We used precleared nulcear extract for ChIP experiment with anti-GFP antibodies (Roche, clones 7.1 and 13.1 mixture). After stringent wash, we performed reverse-crosslinking treatment and purified the ChIP DNA.
We prepared Illumina multiplexed libraries. We prepared 15 (or 20) individual libraries separately (one from each sample) and added a unique barcode to each library at the final step of PCR amplification. We pooled equal amounts of DNA from each library and sequenced one pool per lane of a flow cell on Illumina Genome Analyzer II using the manufacturer's recommended pipeline (versions 1.2 and 1.3, read length = 50 bases)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
CM_CtermGFP_hr01
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| Data processing |
After demultiplexing, the data was mapped to the D. discoideum genome with bowtie version 0.12.7. Read qualities were used. Other parameters: seed length = 28, allowed mismatches = 2, allowed up to 1 alignment per read, with bowtie options -a, --strata, --best. Unmapped reads were trimmed from 3' by 2 nucleotides, which was repeated 5 times.
Peak calling was done on 5-kb genomic regions using partially overlapping (50% overlap), sliding windows of 200 bp. For each window, we calculated the log-ratio of the scaled coverage (scaled by number of mapped reads) between IP+ and control ChIP-seq experiments while permitting a maximum of five mapped reads to share the same genomic coordinates. At each time point, we identified peaks as genomic regions preferentially associated with the TF (GtaC) in the sample compared to control (enrichment > 4-fold; FDR < 0.01; read support > 50). In the proximity of candidate peaks, we accounted for shifts (up to 200 bp) in the distributions of reads mapping to positive and negative strands (Jothi et al. 2008) and enforced similar read support on either strand (absolute log-ratio < 0.5). We also used MACS (Zhang et al. 2008) to identify peaks (parameters: shiftsize=150, bw=200, slocal=5000) and only used peaks identified in both methods. At each time point, ChIP-seq performed on the null strain (carrying GFP), was considered to be the control.
supplementary_files_format_and_content: Processed ChIP-seq files contain the genomic coordinates of predicted peaks and the enrichment of coverage in the IP+ compared to the control ChIP-seq experiments within each region.
Genome_build: D. discoideum Chromosomal DNA: 1,2,3,4,5,6,M, and floating contigs (created: 05-13-2009 13:53) from the DictyBase (chromosomes 1,2,3,4,5,6 and mitochondrial DNA are the same as on NCBI assembly "dicty_2.7"). Regions [3016085, 3768655] from chr2, [64985, 72996] from chrBF and [42801, 78150] from chrR were masked.
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| Submission date |
Nov 10, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Gad Shaulsky |
| E-mail(s) |
gadi@bcm.edu
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| Organization name |
Baylor College of Medicine
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| Department |
Molecular and Human Genetics
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| Street address |
One Baylor Plaza
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL9379 |
| Series (1) |
| GSE63151 |
The GATA transcription factor GtaC regulates early developmental gene expression dynamics in Dictyostelium |
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| Relations |
| BioSample |
SAMN03174651 |
| SRA |
SRX757017 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1542237_CM_CtermGFP_hr01_enrich.txt.gz |
34.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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