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| Status |
Public on Aug 09, 2017 |
| Title |
LELab_ChIP_seq_TLS1637_anti_FLAG |
| Sample type |
SRA |
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| Source name |
Escherichia coli AB1157
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| Organism |
Escherichia coli |
| Characteristics |
developmental stage: mixed population, exponential phase
genotype: AB1157 ybbD::parS site 1 pUTC18::parB (WT)
antibody: Flag
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| Treatment protocol |
T18-ParB (WT) were produced by addition of 0.5mM IPTG for an hour before formadehyde to 1% (final concentration ) was added to fix cells for ChIP-seq
None
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| Growth protocol |
Cultures of Caulobacter (TLS1631-TLS1633) were grown at 30oC in PYE and supplemented with antibiotics, as necessary, at appropriate concentrations. To deplete wild-type non-tagged ParB, exponential-phase cells were washed off xylose and re-introduced to PYE+0.2% glucose for an additional 5 hours. After 4 hours, vanillate was added to induce the expression of flag-parB (WT) or flag-parB (G101S/R104A) for an hour. Cultures of Escherichia coli (TLS1637-TLS1650) were grown at 30oC in LB and supplemented with antibiotics, as necessary, at appropriate concentrations. IPTG (0.5mM) was added to induce the production of T18-ParB (WT) or T18-ParB (G101S). After an hour, formadehyde (1% final concentration) were added to fix cells for ChIP-seq.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the Qiagen Cell Lysis and Protein Precipitation solutions. Detailed protocols are listed in the Supplementary Materials
Standard library construction for Illumina Hiseq2500 sequencing platform
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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 |
For analysis of ChIP-seq data, Hiseq 2500 Illumina short reads (50 bp) were mapped back to the Caulobacter NA1000 reference genome (NCBI Reference Sequence: NC-011916.1) using Bowtie 1 (Langmead et al., 2009) and the following command:bowtie -m 1 -n 1 --best --strata -p 4 --chunkmbs 512 NA1000-bowtie --sam *.fastq > output.sam. Subsequently, the sequencing coverage at each nucleotide position was computed using BEDTools (Quinlan and Hall, 2010) using the following command: bedtools genomecov -d -ibam output.sorted.bam -g NA1000.fna > coverage_output.txt. For analysis of E. coli ChIP-seq data, reference genomes were first reconstructed in silico by inserting the nucleotide sequence of parS and apramycin antibiotic resistance cassette to the ybbD locus of E. coli MG1655 genome. Afterwards, Hiseq 2500 Illumina short reads were mapped back to these reconstructed reference genomes using Bowtie 1. Sequence coverage at each nucleotide position was also computed using BEDTools. Finally, ChIP-seq profiles were plotted with the x-axis representing genomic positions and the y-axis is the number of reads per base pair per million mapped reads (RPBPM) using custom R scripts.
For analysis of IDAP-seq data, Hiseq 2500 Illumina short reads (50 bp) were mapped back to the Caulobacter NA1000 reference genome (NCBI Reference Sequence: NC-011916.1) using Bowtie 1 (Langmead et al., 2009) and the following command:bowtie -m 1 -n 1 --best --strata -p 4 --chunkmbs 512 NA1000-bowtie --sam *.fastq > output.sam. Subsequently, sequencing reads were sorted to either being mapped to the upper DNA strand or to the lower strand of the reference genome, as suggested in the original IDAP-seq publication (Belitsky and Sonenshein, 2013). The number of 5’ end of reads that were mapped to the upper strand was counted for each nucleotide position along the Caulobacter genome using BEDTools (Quinlan & Hall, 2010) and the following command: bedtools genomecov -d -5 -strand + -ibam output.sorted.bam -g NA1000.fna > upper_strand_output.txt. To count the number of 5’ end of reads that were mapped to the lower strand, the following command was used instead: bedtools genomecov -d -5 -strand - -ibam output.sorted.bam -g NA1000.fna > lower_strand_output.txt. The IDAP-seq profile was then plotted using R. The sequence in between the summit of upper strand profile and that of the lower strand profile defines the minimal parS sequence required for binding to ParB.
Hiseq 2500 Illumina short reads (50 bp) were mapped back to the Caulobacter NA1000 reference genome (NCBI Reference Sequence: NC-011916.1) using Bowtie 1 (Langmead et al., 2009) and the following command: bowtie -m 1 -n 1 --best --strata -p 4 --chunkmbs 512 NA1000-bowtie --sam *.fastq > output.sam. Subsequently, the sequencing coverage for each nucleotide position was computed using BEDTools (Quinlan & Hall, 2010) and the following command: bedtools genomecov -d -ibam output.sorted.bam -g NA1000.fna > coverage_output.txt. Finally, the ratio between the number of reads of libraries generated from pMCS1-Tn5-ME-R6Kγ-kanR-ME or pMCS1-Tn5-ME-R6Kγ-kanR-parS456-ME were calculated. Results were binned over 1 kb and represented as a log10 scale.
Genome_build: NC_000913.3
Genome_build: NC_011916.1
Supplementary_files_format_and_content: Files ending with _coverage.txt: tab-delimited text file of nucleotide-resolution coverage from RNA-seq data (column 1: genome ID, column 2: nucleotide position, column 3: coverage)
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| Submission date |
Jun 20, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Tung Ba Khanh Le |
| E-mail(s) |
tung.le@jic.ac.uk
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| Phone |
01603450776
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| Organization name |
John Innes Centre
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| Department |
Department of Molecular Microbiology
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| Lab |
www.tunglelab.org
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| Street address |
Colney Lane
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| City |
Norwich |
| State/province |
Norfolk |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
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| Platform ID |
GPL18133 |
| Series (1) |
| GSE100233 |
Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome |
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| Relations |
| BioSample |
SAMN07257845 |
| SRA |
SRX2935304 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2675514_LELab_ChIP_seq_TLS1637_anti_FLAG_coverage.txt.gz |
14.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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