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Sample GSM4290266

Query DataSets for GSM4290266

Status

Public on Mar 19, 2020

Title

RpoABC_ChIP-Seq_WT(MB1)_(AIYT-161)

Sample type

SRA

Source name

bacterial culture

Organism

Caulobacter vibrioides NA1000

Characteristics

strain: NA1000
genotype: wild-type
chip antibody: RNA polymerase antibody sampler kit (Cat 69907, Biolegend)

Treatment protocol

C. crescentus wildtype and derivative cells were treated with 1% formaldehyde in 1 μM sodium phosphate buffer (pH 7.6) at RT for 10 min to achieve crosslinking. Subsequently, the cultures were incubated for an additional 30 min on ice and washed three times in phosphate buffered saline (PBS, pH 7.4). The resulting cell pellets were stored at -80°C.

Growth protocol

Overnight saturated cultures of Caulobacter crescentus strains were freshly diluted in PYE media contaning 0.3% xylose and cultures were grown at 30°C to mid-log phase (OD600nm~0.5).

Extracted molecule

genomic DNA

Extraction protocol

After resuspension of the cells in TES buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl) containing 10 mM of DTT, they were incubated in the presence of Ready-Lyse lysozyme solution (Epicentre, Madison, WI) for 10 minutes at 37°C, according to the manufacturer's instructions. Lysates were sonicated (Bioruptor® Pico) at 4°C using 15 bursts of 30 sec to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared by centrifugation at 14,000 rpm for 2 min at 4°C. The volume of the lysates was then adjusted (relative to the protein concentration) to 1 ml using ChIP buffer (0.01% SDS, 1.1% Triton X-84 100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl) containing protease inhibitors (Roche) and pre-cleared with 80 μl of protein-G agarose (Roche, www.roche.com) and 100 μg BSA. The rest of the pre-cleared lysates was incubated overnight at 4°C with E. coli RNA polymerase antibody sampler kit (Cat 699907, Biolegend https://www.biolegend.com/fr-ch/products/e--coli-rna-polymerase-antibody-sampler-kit-15078) with the different antibodies mixed with a ratio of 1:1:1 (1:500 dilution). The immuno-complexes were captured after incubation with Protein-G agarose (pre-saturated with BSA) during a 2 h incubation at 4°C and they were washed subsequently with low salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), with high salt washing buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), with LiCl washing buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and finally twice with TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). The complexes were eluted from the Protein-G agarose beads with two times 250 μl elution buffer (SDS 1%, 0.1 M NaHCO3, freshly prepared) and then, incubated overnight with 300 mM NaCl at 65°C to reverse the crosslinks. The samples were then treated with 2 μg of Proteinase K for 2 h at 45°C in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5). DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1), ethanol-precipitated using 20 μg of glycogen as a carrier and resuspended in 50 μl of DNAse/RNAse free water. Immunoprecipitated chromatin was used to prepare sample libraries used for deep-sequencing at Fasteris SA (Geneva, Switzerland). ChIP-Seq libraries were prepared using the DNA Sample Prep Kit (Illumina) following manufacturer instructions.
TruSeq ChIP Library Preparation Kit

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Description

AIYT161-162-163 20bp.txt
AIYT161-162-163 G1 gene.txt
AIYT161-162-163 S gene.txt

Data processing

basecalling: HiSeq Control Software HD 3.4.0.38; RTA 2.7.7; bcl2fastq2.17 v2.17.1.14
Alignement: map with BWA (Galaxy Version 0.7.17.4)
quantification: SeqMonk software 1.40.0 (Braham Bionformatics Institute)
Genome_build: Caulobacter crescentus NA1000 NC_011916.1
Supplementary_files_format_and_content: txt files were generated using annotated probe report from SeqMonk. The AIYT-161-162-163 20bp was generated using 20bp probe all along the chromosome. The AIYT-161-162-163 G1 gene and AIYT-161-162-163 S gene represent specifically the promoter region of the G1 and S regulon of CtrA based from Fumeaux et al (PMID: 24939058) and Schrader et al (PMID: 25078267). In this case, probe of 200bp before and after the beginning of the CtrA regulated gene were created to quantify the occupancy of the polymerase on the CtrA target gene. The sheet is organized in column as follow: column 1: chromosome name; colum 2: start of the probe; column 3: end of the probe; column 4: feature; column 5: strand of the gene; column 6: reads in RPM for WT (AIYT161); column 7: reads in RPM for ∆citA (AIYT162); column 8: reads in RPM for xylX::relA' (AIYT163)

Submission date

Jan 30, 2020

Last update date

Mar 19, 2020

Contact name

Patrick Viollier

E-mail(s)

patrick.viollier@unige.ch

Organization name

University of Geneva

Street address

Rue michel Servet,1