|
| Status |
Public on Aug 11, 2017 |
| Title |
Input Mock |
| Sample type |
SRA |
| |
|
| Source name |
Asynchronous population
|
| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
strain: NA1000
genotype: xylX::pXVENC4 (CJW5796)
expression: Venus fluorescent protein at gene locus expressed at xylose locus, induced 1h30min xylose 0.03% before fixation
|
| Treatment protocol |
DNA was cross-linked to proteins with addition of 1% formaldehyde in presence of 7.5mM Sodium phosphate pH 7,6. Incubated at room temperature for 10 min and 30min on ice. Cells were collected by centrifugation at 7100 xg rpm for 10 min at 4C. The supernatant was discarded and the pellet washed twice with 20mL PBS buffer, pellet was kept at 4C and used immediately for DNA extraction.
|
| Growth protocol |
ChIP-seq samples were obtained from mid-log (OD660nm ≈ 0.4) cultures of CJW5534 producing RedN-Venus grown in PYE at 30°C. In parallel, a “mock ChIP” sample was collected from a PYE culture of CJW5796 producing freely diffusing Venus after 1h 30 min of induction with 0.03% xylose.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immunoprecipitated DNA was extracted as described before (Fumeaux et al., 2014) with the following modifications: PierceTM Protein A/G Magnetic Beads (Thermo Fisher Scientific) and anti-GFP (JL-8) Living Colors® Av Monoclonal Antibody (Clontech Laboratories) were used for protein immunoprecipitation, SDS was excluded from ChIP buffer, Herring sperm DNA was not used to saturate beads and sonication was performed using a Digital Sonifier® S-250D on ice with the 1/8” microtip (Branson Sonic Power Co) with 45% output and 10 cycles of 30 s ON/30 s OFF.
Quality and concentration of the ChIP-DNA was assessed by estimating the A260/A280 and A260/A230 ratios with a Nanodrop device (Thermo Scientific). Library preparation and sequencing was performed by the Yale Center for Genome Analysis as follows: ~10ng of immunoprecipitated DNA was evaluated with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA) using a Quant-iT™ DNA Assay Kit high sensitivity (Thermo Fisher Scientific). ChIP-DNA was further purified using Ampure XP SPRI beads (Beckman Coulter Genomics). The DNA was then end-repaired, A-tailed, adapter ligated and PCR enriched (8 -10 cycles). Indexed libraries that met appropriate cut-offs were quantified by both qRT-PCR and insert size distribution determined with the LabChip GX. Samples with a yield of ≥0.5 ng/ul are used for sequencing. Subsequently, for sample concentrations were normalized to 10 nM and loaded into Illumina Rapid or High-output flow cells (Illumina®, San Diego CA) at a concentration that yields 150-250 million passing filter clusters per lane. Samples were sequenced using 75 bp single or paired-end sequencing on an Illumina HiSeq 2500 according to Illumina protocols. The 6 bp index was read during an additional sequencing read that automatically follows the completion of read 1. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ControlG2
|
| Data processing |
The reads from the sequencer were trimmed using FastX Trimmer.
The reads were aligned to the Caulobacter crescentus (NA1000) strain. This reference strain was downloaded from the NCBI Refseq and indexed using bwa. The reads were alined to this index using bwa mem
Perbase coverages were calculated using coverageBed program, and these values were further normalized to million mapped reads.
Genome_build: Caulobacter crescentus (NA1000)
Supplementary_files_format_and_content: The processed files are the normalized coverage files in BEDGRAPH format. The first column denotes the chromosome, the second and their column denote the span, and the last column denotes the normalized coverage per million mapped reads.
|
| |
|
| Submission date |
Aug 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Christine Jacobs-Wagner |
| E-mail(s) |
christine.jacobs-wagner@yale.edu
|
| Phone |
203-737-6778
|
| Organization name |
Yale University
|
| Department |
Molecular, Cellular and Developmental Biology
|
| Lab |
Microbial Sciences Institute, Jacobs-Wagner Lab
|
| Street address |
840 West Campus Drive, PO BOX 27388
|
| City |
West Haven |
| State/province |
CT |
| ZIP/Postal code |
06516 |
| Country |
USA |
| |
|
| Platform ID |
GPL18276 |
| Series (1) |
| GSE85344 |
ChIP-seq data of RedN-venus from asynchronous and swarmer populations of Caulobacter crescentus NA1000 |
|
| Relations |
| BioSample |
SAMN05526401 |
| SRA |
SRX2009088 |
