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| Status |
Public on May 13, 2015 |
| Title |
anti-FLAG sample |
| Sample type |
SRA |
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| Source name |
LB liquid medium planktonic cells
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| Organism |
Vibrio cholerae |
| Characteristics |
strain: C7258
od600 nm: 0.5
phenotype: C7258 hns::hns-flag
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| Growth protocol |
V. cholerae cells were grown in 50 mL LB medium at 37°C with agitation (225 rpm) to optical densities at 600 nm (OD600) of 0.5. Culture media was supplemented with ampicillin (Amp, 100 µg/mL).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The ChIP protocol was carried out as described before (Wang H., et al. 2012. Appl Environ. Microbiol. 78(7):2482-8) with modifications. Briefly, nine 100 µl cell lysate samples were combined to perform the immunoprecipitation. The final purified IP DNA was resuspended in 30 µl for a concentration factor of 60 compare to our regular ChIP procedure. The DNA concentration of the anti-FLAG (7.2 ng/µl) and input (23.0 ng/µl) samples were determined by the Qubit® Fluorometric Quantitation System (Life Technologies).
Twenty ng of anti-FLAG and input DNA samples were used for library preparation for the Illumina sequencing using the TruSeq ChIP kit (Illumina, San Diego, CA) following manufacturer’s protocol. Briefly, DNA samples were end-repaired and phosphorylated using a combination of Klenow, T4 DNA Polymerase and T4 polynucleotide kinase. An A base was added to the newly blunt ended molecules followed by adaptor ligation is a standard ligation reaction. Utilizing adaptors than contain a 6 bp index it was run several samples per lane in the HiSeq2000 instrument (Illumina). The ChIP-Seq libraries were then amplified using PCR, quantitated with qPCR and loaded to a standard concentration of 2.5 pM per lane. A standard paired-end sequencing reaction was done generating 50 bp of sequence from each direction of sequencing. The raw data was converted from .bcl file format to .fastq file format for downstream analysis. This was done using CASAVA v1.8.2 software from Illumina.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The raw fastq data files were aligned to the V. cholerae EL TOR N16961 reference genome (NC_002505.1 and NC_002506.1) using Bowtie, an ultrafast, memory-efficient short read aligner available on the Galaxy web-based platform. The generated sam data files were further filtered out to remove unmapped reads and converted to bam data files using the Galaxy NGS: SAM Tools. For H-NS binding sites (peaks) definition and quantitation, peak annotation reports and location of the nearest downstream genes, SeqMonk v0.27.0 was used, a program for visualizing and analyzing large set of mapped genomic regions (Babraham Institute). For peak calling, the Model-based Analysis of ChIP-Seq (MACS) algorithm within SeqMonk was used adjusting the P-value cutoff to 1.0E-05, the sonicated fragment size to 300 bp and using the input sample as a control. For peak quantitation the Read Count Quantitation panel was used correcting for total counts and log-transforming the counts.
Genome_build: NCBI Reference Sequences: NC_002505.1 and NC_002506.1
Supplementary_files_format_and_content: The excel file contains a report of the H-NS peaks detected by the peak calling analysis. It contains the genome coordinates of the peaks, the locus tag of the gene at the beginning of the peaks, the H-NS mean enrichment of the peaks, and the function associated with genes inside the peaks. The bed files contain the genome coordinates of H-NS peaks and the coordinates at the summit of the peaks. The wiggle plot files contain the wiggle plots for the Anti-FLAG and Input samples
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| Submission date |
Dec 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
JULIO CESAR AYALA |
| E-mail(s) |
jayalaf@emory.edu
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| Phone |
404-321-6111
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| Organization name |
Emory University
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| Department |
MICROBIOLOGY AND IMMUNOLOGY
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| Lab |
William Shafer
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| Street address |
1670 Clairmont Rd VA/MC 5A188
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| City |
DECATUR |
| State/province |
GA |
| ZIP/Postal code |
30033 |
| Country |
USA |
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| Platform ID |
GPL19265 |
| Series (1) |
| GSE64249 |
Genome-wide DNA-binding profile of the Vibrio cholerae histone-like nucleoid structuring protein (H-NS) |
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| Relations |
| BioSample |
SAMN03266809 |
| SRA |
SRX814210 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1567051_H-NS-peaks.bed.gz |
1.1 Kb |
(ftp)(http) |
BED |
| GSM1567051_H-NS-peaks_summit.bed.gz |
1.0 Kb |
(ftp)(http) |
BED |
| GSM1567051_Wiggle_plot_Chr-1_.wig.gz |
941.0 Kb |
(ftp)(http) |
WIG |
| GSM1567051_Wiggle_plot_Chr-2_.wig.gz |
340.8 Kb |
(ftp)(http) |
WIG |
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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