characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2017-08-24T17:01:41Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104062012
characteristics.characteristic: External Id
characteristics.text: unspecified
characteristics.characteristic: INSDC center name
characteristics.text: 2017-08-24T17:01:41Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-05-16T18:32:04Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1725905
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-5745:rccR_Pyr_Tot
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: Protocols: P. fluorescens strains were grown at 28 degree Celsius in lysogenic broth. After bacterial growth, formaldehyde (1%) was added to the cultures and incubated at room temperature for 20 min, before the reaction was quenched with glycine (125 mM) for 5 min. Cells were collected and washed with cold PBS four times. The cells were lysed in 1 ml of lysis buffer solution (10 mM Tris-HCl pH8, 50 mM NaCl, 4 mg/ml lysozyme, 1x protease inhibitor). Cell extracts were then sonicated to fragment the DNA to an average size of 500bp. 50 µl of the extract was removed for total DNA preparation. For immunoprecipitation of RccR cross-linked DNA, a portion of the extract (1 ml) was immunoprecipitated with 10 µl of polyclonal anti-RccR antibody at 4 °C for 4h. After incubation with ProteinA affinity gel (Sigma) for 1h at 4 °C, the beads were washed twice with IP buffer (100 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.5% Triton X-100, 0,1 % SDS, 1x protease inhibitor), and finally with TE buffer. The immunoprecipitated material was eluted with 100 µl of elution buffer (50 mM Tris-Cl pH 7.6, 10 mM EDTA, 1% SDS). Cross-linking of immunoprecipitated and total DNA was reversed by incubation at 65°C overnight. After Proteinase K (Roche) treatment, the immunoprecipitate and total DNA samples were extracted using phenol-chloroform and purified using a QiaQuick kit (Qiagen). Illumina TruSeq ChIP Library Preparation Kit was used to prepare the libraries which were then size-selected to 200 to 300 base pairs.
characteristics.characteristic: description
characteristics.text: rccR deletion
characteristics.characteristic: genotype
characteristics.text: M9 minimal medium with pyruvate
characteristics.characteristic: growth condition
characteristics.text: Pseudomonas fluorescens
characteristics.characteristic: organism
characteristics.text: Pseudomonas fluorescens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_294
characteristics.characteristic: organism
characteristics.text: Pseudomonas fluorescens
characteristics.characteristic: scientific_name
characteristics.text: rccR_Pyr_Tot
characteristics.characteristic: title