Illumina HiSeq 4000 sequencing; Chip-seq to identify HrdB regulated genes in Streptomyces coelicolor
Chip-seq to identify HrdB regulated genes in Streptomyces coelicolor
Spores stocks of Streptomyces coelicolor A(3)2 M145 were prepared by harvesting spores from agar plates grown for 10 days. Following the procedure described in Nieselt et al.[Nieselt, 2010], we inoculated thawed spore stock (2,5*10^9/ml CFU – typicaly 3 ml) to 100ml 2YT medium (bacto tryptone, 16 g/l; bacto-yeast extract, 10g/l; NaCl, 5g/l) to 500ml Erlenmeyer's flask (customised to enable aeration) and germinated at 30°C for 5 hours. The germinated spores were harvested by centrifugation (3200 × g, 15°C, 5 min) and resuspended in 5 ml ion-free water. Then they were inoculated into an 1.8l Na-glutamate medium (Na-glutamate monohydrate, 61 g/l; glucose monohydrate, 44 g/l; MgSO4, 2.0 mM; Na2HPO4, 2.3 mM; KH2PO4, 2.3 mM) supplemented with 8ml/l of trace element solution (ZnSO4 x 7 H2O, 0.1 g/l; FeSO4 × 7 H2O, 0.1 g/l; MnCl2 × 4 H2O, 0.1 g/l, CaCl2x6H2O,0,1g/l, NaCl 0,1g/l ) [Kieser, 2000] and 5.6 ml/l of TMS1 (FeSO4 × 7 H2O, 5 g/l; CuSO4 × 5 H2O, 390 mg/l; ZnSO4 × 7H2O, 440 mg/l; MnSO4 × H2O, 150 mg/l; Na2MoO4 × 2 H2O, 10 mg/l; CoCl2 × 6 H2O, 20 mg/l; HCl, 50ml/l). Cultivation was conducted at 30 °C at 250 RPM and pH of 7 was maintained during growth. ChIP-seq analysis was done according to [Spencer, 2003] with minor modifications. After 22 hours of growth, cells were incubated for 30 minutes with 1% formaldehyde (CH2O 36,5%-38% Sigma Aldrich F8775). Cross-link reactions were stopped with pre-chilled 2 M glycine for 5 minutes at room temperature. Cells were harvested by centrifugation at 4 °C and washed five times with 1xPBS.  The pellet was stored at -80°C prior further processing. Subsequently, the pellet was resuspended in RIPA buffer (SDS, 0.1%; Sodium deoxycholate, 0.5%; Triton X-100, 0.5%; NaCl 150mM; Tris, pH 8, 50 mM;  Proteases inhibitor-Complete, Mini, EDTA-free, Roche, 10mM) and sonicated 6 x 15-s, amplitude 0.5, 1 cycle on ice (Hielscher sonicator, UP 200S). The cell lysate was centrifuged at 4 °C, 20 000 x g, 20 minutes . Supernatant was collected and precleared for 2 hours at 4 °C by pre-equilibrated Protein-G Plus Agarose (Santa Cruz sc2002) in RIPA buffer. The concentration of proteins was measured and 2 mg of total proteins in lysate were added to 2 µg of anti-HA high affinity antibody (Roche, 11867423001) and incubated for 16 hours at 4 °C. Protein-G Plus Agarose was added and incubated for 6 hours at 4 °C. Samples were washed 3 times with RIPA buffer, 4 times with WASH buffer (Triton X-100, 0.5%; Sodium deoxycholate, 0.4%; LiCl, 0.5 M; Tris, pH 8.5, 100 mM) twice with RIPA buffer a second time, twice with TE buffer (EDTA, 10 mM; Tris-HCl, pH 8, 10 mM). Subsequently, elution was achieved using Elution buffer (SDS, 1%; EDTA, 10 mM; Tris-HCl, pH 8, 50 mM). Decrosslink reactions were done by addition of 200 mM NaCl and Proteinase K at 60 °C overnight. Finally, DNA was purified by NucleoSpin gDNA Clean-up (Macherey-Nagel). Library construction and sequencing where outsourced to GeneCore sequencing facility in EMBL Heidelberg. Following is protocol provided by the sequencing facility: Libraries were prepared with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB #E7645), and all material was used as input. The adaptors were diluted 1:30 times, and 17 PCR cycles were performed.  Libraries were measured with Qubit DNA High sensitivity assay, and afterwards loaded on Agilent Bioanalyzer 2100 with DNA 1000 kit.  Samples were pooled equimolar before sequencing, and the pool was measured with Qubit DNA high sensitivity kit.
Experimental Factor: immunoprecipitate
anti-HA
Experimental Factor: genotype
wild type genotype