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Sample GSM3963019 Query DataSets for GSM3963019
Status Public on Jul 22, 2020
Title LeLab_ChIP_seq_MT148_vanA_Pvan_1xFLAG_yfp_rep1
Sample type SRA
 
Source name Caulobacter crescentus NA1000
Organism Caulobacter vibrioides NA1000
Characteristics genotype: MT148 (parB::Pxyl-parB)+ vanA::Pvan-1xFLAG-yfp
developmental stage: mixed population, exponential phase
chip-seq antibody: FLAG
Treatment protocol FLAG-ParB were produced in medium supplemented with 0.3 % glucose and 0.5 mM vanillate before formaldehyde to 1% (final concentration) was added to fix cells for ChIP-seq.
Growth protocol For E. coli ChIP-seq, cells harboring pUT18C-1xFLAG-ParB were grown in LB (25 mL) at 28oC to mid-exponential phase (OD600 ~0.4) before 1 mM IPTG was added for 1-3 hours. The induction time (either 1, 2 or 3 hours) was chosen so that all ParB variants were produced to a similar protein level as judged by an α-FLAG immunoblot. Subsequently, formaldehyde was added to a final concentration of 1% to fix the cells. For C. crescentus ChIP-seq, MT148 cells harboring van::Pvan-FLAG-ParB (WT)/mutants were grown in PYE (25 mL) + 0.3% glucose + 0.5 mM vanillate to mid-exponential phase (OD600 ~0.4) before formaldehyde was added to a final concentration of 1% to fix the cells.
Extracted molecule genomic DNA
Extraction protocol For ChIP-seq, DNA was isolated using Qiagen PCR purification columns.
For ChIP-seq libraries, a standard library construction procedure (using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina sequencing) was employed.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
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 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 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, the same procedure as above was applied, except that short reads were map to the reference genome of the E. coli MG1655 (NCBI Reference Sequence: NC_000913.3). 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.
Genome_build: NC_011916.1 (C. crescentus)
Genome_build: NC_000913.3 (E. coli)
Supplementary_files_format_and_content: *_coverage_output.txt: The processed data files for ChIP-seq are tab-delimited files containing three fields: the genome build, the nucleotide position, and the coverage at that position.
 
Submission date Jul 22, 2019
Last update date Jul 22, 2020
Contact name Tung Ba Khanh Le
E-mail(s) tung.le@jic.ac.uk
Phone 01603450776
Organization name John Innes Centre
Department Department of Molecular Microbiology
Lab www.tunglelab.org
Street address Colney Lane
City Norwich
State/province Norfolk
ZIP/Postal code NR4 7UH
Country United Kingdom
 
Platform ID GPL26943
Series (1)
GSE134665 Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation
Relations
BioSample SAMN12338882
SRA SRX6488018

Supplementary file Size Download File type/resource
GSM3963019_LeLab_ChIP_seq_MT148_vanA_Pvan_1xFLAG_yfp_rep1_coverage_output.txt.bz2 10.7 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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