M600-ip
Streptomyces coelicolor A3(2)
Protocols: Streptomyces coelicolor A3(2) M600 (NCBI Ref: NC_003888) was used as the wild type strain. A PCR-based approach was used to generate an internal in-frame deletion in sigE. This strain was named J2130. Full details are in Paget et al 1999 J Bacteriol. Jan;181(1):204-11. 3xFLAG tagged sigE was created via a two step fusion-PCR and cloned in the plasmid pMS82 which was then introduced into J2130 by conjugation using the dam dcm hsdS E. coli strain ET12567 containing pUZ8002. Spores(equivalent to an initial OD450≈ 0.025 in the final culture) were germinated by heat shock treatment in 5 ml of TES buffer (0.05 M, pH 8) at 50°C for 10 min, then diluted with an equal volume of double‐strength germination medium [1% (w/v) Difco yeast extract, 1% (w/v) Difco casamino acids, 0.01 M CaCl2] and incubated with shaking at 37°C for 2-3 h. Germinated spores were inoculated into NMMP and grown to mid-log phase (OD600 approx 0.5) at 30°C. At OD 450 ~ 0.6, vancomycin was added to a final concentration of 10 μg/ml and samples were collected at 15 min intervals for 1 hour. For each of M600 and SCH06 the enrichment ratios of the total (non-IP) DNA were subtracted from those of IP DNA. The resulting differences (two files) were used for calculating P-values assuming normal distribution. Finally, the P-values were adjusted using the Benjamini and Hochberg method as available in the p.adjust function of R. Genomic positions where SCH06 had adjusted P-values <= 1e-4 and M600 had adjusted P-values > 1e-4 were considered to be binding sites for SigE. Context of genes around such sites was computed using Perl script and reported in the file gene_context_top_sig.txt. Formaldehyde was added to cultures at a final concentration of 1% (vol/vol), and incubation was continued for 30 min. Glycine was then added to a final concentration of 125 mM to stop the cross-linking. The samples were left at room temperature for 5 min and washed twice in phosphate-buffered saline (PBS) buffer (pH 7.4). The pellets were resuspended in 0.5 ml of lysis buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl), containing 15 mg ml−1 of lysozyme and 1× protease inhibitor (Roche Applied Science) and incubated at 37°C for 25 min or until lysed. Subsequently, 0.5 ml immunoprecipitation (IP) buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 0.8% [vol/vol] Triton X-100), containing 1× protease inhibitor, was added, and the samples were chilled on ice. The samples were sonicated for six cycles of 20 s each at 10 µm to shear chromosomal DNA into fragments ranging from 300 to 1,000 bp on average. The samples were centrifuged twice at 16,000 × g at 4°C for 10 min to clear the cell extracts, after which 50 µl of each was set aside for total-DNA extraction. The remaining lysates were added to M2 (Sigma-Aldrich A2220) gel suspension (40 µl per ml of lysate), prepared and washed as described in the manufacturer's instructions. The mixtures were then incubated on a rotating wheel at 4°C overnight. The samples were centrifuged for 30 s at 4°C and 5,500 × g, and the pellets were washed twice with 0.5× IP buffer and then twice with 1× IP buffer and transferred to new Eppendorf tubes after the first washing step. The pellets and 50 µl of total cell extracts (set aside earlier) were eluted overnight at 65°C in 100 µl of IP elution buffer (50 mM Tris-Cl, pH 7.6, 10 mM EDTA, 1% SDS) to reverse the cross-links. The samples were centrifuged at 16,000 × g for 5 min to remove the beads. The pellets were re-extracted with 50 µl of TE buffer (10 mM Tris-Cl, pH 7.4, 1 mM EDTA) and incubated with 0.2 mg ml−1 proteinase K (Roche) for 2 h at 55°C. The samples were extracted twice with phenol-chloroform and once with chloroform and further purified using QiaQuick columns (Qiagen). DNA was eluted in 50 µl EB buffer (10 mM Tris-Cl, pH 8.5) and quantified using a NanoDrop spectrophotometer (Thermo Scientific). Library construction and sequencing were performed using the TruSeq ChIP sample preparation kit from Illumina Inc. (part 15023092, revision A, August 2012) according to the recommended protocol.
ENA-FIRST-PUBLIC
2019-04-24T17:04:15Z
ENA-LAST-UPDATE
2019-03-27T10:44:44Z
External Id
SAMEA5537688
INSDC center name
Department of Molecular Microbiology John Innes Centre
INSDC first public
2019-04-24T17:04:15Z
INSDC last update
2019-03-27T10:44:44Z
INSDC status
public
Submitter Id
E-MTAB-7827:M600-ip
broker name
ArrayExpress
genotype
wild type genotype
sample name
E-MTAB-7827:M600-ip
scientific_name
Streptomyces coelicolor A3(2)
strain
M600