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Sample GSM2830260

Query DataSets for GSM2830260

Status

Public on Oct 26, 2017

Title

54_3; Native Cmr, basal cAMP levels

Sample type

SRA

Source name

Broth culture of M. bovis BCG WT, Ambient (21% O2, 0% CO2) shaking, 7 days

Organism

Mycobacterium tuberculosis variant bovis BCG str. Pasteur 1173P2

Characteristics

chip antibody: Anti-Cmr antiserum
transcription factor expression level: Native Cmr
camp treatment/level: Basal

Treatment protocol

All bacterial cultures were grown under the mentioned O2 and CO2 conditions for 7 days (mid-log phase of growth). For exogenous cAMP treatment, 10mM dibutyryl-cAMP was added to the bacterial cutlure on day 3 and returned to the controlled atmospheric incubator. dbcAMP is a cell permeable compound, which gets cleaved upon internalization by bacteria to produce cAMP. For endogenous cAMP supplementation, M. bovis BCG (p0805:Rv1264cat) strain was used. In this strain, constitutive expression of the catalytic domain of Rv1264 adenylyl cyclase, driven by the Rv0805 promoter, increases the cAMP level within the bacterial cytoplasm.

Growth protocol

Liquid cultures of all strains used- M. bovis BCG WT, M. bovis BCG (p0805:Rv1264cat), M. bovis BCG (pMBC357) were started from frozen seed stocks and grown shaking for 7 days in mycomedium, with gentle rocking in 225-cm2 tissue culture flasks at a shallow depth of 2 mm. Bacterial cultures were either grown in ambient air conditions (21% O2) or in CO2 supplemented low oxygen conditions (1.3% O2, 5% CO2) in a controlled atmospher incubator.

Extracted molecule

genomic DNA

Extraction protocol

A total of 50 ml culture volume of mid-log phase bacterial culture was cross-linked with 1% formaldehyde, for 30 min at room temperature, on a slow rocking platform. Cross-linking was quenched by addition of 250mM glycine and incubation for 15 min at room temperature with slow rocking. The cells were harvested, washed with cold PBS and resuspended in 0.5-0.6 ml of Buffer I (20 mM HEPES pH 7.9, 50 mM KCl, 0.5 M DTT, 10% glycerol) with protease inhibitor cocktail (Sigma). Bacterial cells were lysed and DNA sheared by sonication for 25 min (Covaris). The salt concentration of the cleared cell lysate was adjusted to a final concentration of 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40 (IPP150 buffer). Immunoprecipitation was carried out by incubation of lysate with 10ul of Cmr antiserum, on nutator at 4°C. A total of 50ul of Protein A agarose beads were rinsed with IPP150 buffer, and added to the lysate-antiserum mixture. Protein A agarose-lysate-antiserum mixture was incubated at 4°C for 30 min and room temperature for 1.5 h. The beads were washed at least five times with IPP150 buffer followed by two washes with TE buffer (10mMTris HCl pH 8.0, 1mM EDTA).DNA from agarose beads was eluted by incubation with 150ul elution buffer (50 mMTris HCl pH 8.0, 10mM EDTA, 1% SDS) at 65°C for 15 min. A second elution was carried out by incubation of pellet with 100ul of TE buffer+ 1% SDS at 65°C for 5 min. Both elutions were pooled and de-crosslinked by incubating with 1 mg/ml Proteinase K at 37°C for 2 h and 65°C overnight. DNA was purified using PCR purification kit (QIAGEN).
Standard manufacturer (Illumina) recommended protocol was used for library construction

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Description

54_3_64Y9CAAXX_030612_ChIP_448_BCG
processed data file:
Cmr Binding sites_GEO Sub.txt

Data processing

Sequencing was performed on the Illumina platform, using a GAIIx (Boston University, sequencing core).
Coverage along the genome was calculated using Bowtie2 and SamTools.
Enriched regions were called using log-normal distributions.
The criteria for a region to be considered ‘enriched’ were a minimum region length of 150 nt and at least 60 nt shift between its forward and reverse peaks.
Region coverage was normalized using mean coverage of an experiment, correcting for the number of reads amongst experiments
Genome_build: M. bovis BCG Pasteur
Supplementary_files_format_and_content: Analyzed data is provided as text files. Sample Name; BCG_start and BCG_Stop are the co-ordinates of the binding site mapped to the M. bovis BCG Pasteur genome; enrichment of a particular region is indicated by the peak height; Site type indicates whether the binding site maps to region between genes (intergenic) or within open reading framr of annotated genes (genic)

Submission date

Oct 25, 2017

Last update date

May 15, 2019

Contact name

Kathleen McDonough

E-mail(s)

[email protected]

Organization name

Wadsworth Center, NYSDOH

Street address

120 New Scotland Ave