|
| Status |
Public on Jul 22, 2020 |
| Title |
LeLab_ChIP_seq_MT148_vanA_Pvan_1xFLAG_Caulobacter_crescentus_ParB_5K_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Caulobacter crescentus NA1000
|
| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
genotype: MT148 (parB::Pxyl-parB)+ vanA::Pvan-1xFLAG-Caulobacter_crescentus_ParB_5K
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 |
SAMN12338845 |
| SRA |
SRX6488015 |
