Illumina HiSeq 2000 sequencing; Role of RccR in the transcriptional control of primary carbon metabolism in Pseudomonas
Role of RccR in the transcriptional control of primary carbon metabolism in Pseudomonas
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.