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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
Phone 01603450776
Organization name John Innes Centre
Department Department of Molecular Microbiology
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
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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