C4
Streptomyces coelicolor A3(2)
Protocols: Spores stocks of Streptomyces coelicolor A(3)2 M145 were prepared by harvesting spores from agar plates grown for 10 days. Streptomyces strains were grown following protocols described previously by Nieselt et al. [PMID: 20053288]. Briefly, spores were prepared by harvesting them from agar plates grown for 10 days. Subsequently, spore germination was conducted at 30 °C for 5 h by inoculation of spores (2,5*10∧9/ml CFU) into 2YT medium (bacto tryptone, 16 g/l; bacto yeast extract, 10 g/l; NaCl, 5 g/l). After germination, culture was washed by 5 ml of ion-free water and inoculated into Na-glutamate medium (Na-glutamate, 61 g/l; glucose monohydrate, 44 g/l; MgSO4, 2.0 mM; Na2HPO4, 2.3 mM; KH2PO4, 2.3 mM) supplemented with 8 ml/l of trace element solution (ZnSO4 × 7H2O, 0.1 g/l; FeSO4 × 7H2O, 0.1 g/l; MnCl2 × 4H2O, 0.1 g/l, CaCl2 × 6H2O,0,1 g/l, NaCl 0,1 g/l) and 5.6 ml/l of TMS1 (FeSO4 × 7H2O, 5 g/l; CuSO4 × 5H2O, 390 mg/l; ZnSO4 × 7H2O, 440 mg/l; MnSO4 × H2O, 150 mg/l; Na2MoO4 × 2H2O, 10 mg/l; CoCl2 × 6 H2O, 20 mg/l; HCl, 50ml/l) [Kieser 2000]. Bacteria were shaken for 23 hours (corresponding to exponential phase Pospíšil et al. [PMID: 33325652]) at 30 °C, 250 rpm and pH 7 was maintained during the growth. The growth continued for 1 hour. After stress treatment we followed by 30 minutes long crosslink reaction using 1% formaldehyde (CH2O 36,5 %–38 % Sigma Aldrich). Crosslinking was stopped with prechilled 2 M glycine for 5 minutes at room temperature. Cells were centrifuged at 4 °C and washed five times with 1×PBS. The pellet was resuspended in RIPA buffer (SDS, 0.1 %; sodium deoxycholate, 0.5 %; Triton X-100, 0.5 %; 1 mM; NaCl, 150 mM; Tris, pH 8, 50 mM; Proteases inhibitor-Complete, Mini, ethylenediaminetetraacetic acid (EDTA)-free, Roche, 10 nM) and sonicated 6×15-s, 0.5 amplitude, on ice (Hielscher sonicator, UP 200S). Lengths of DNA fragments were analyzed by agarose gel electrophoresis (range of DNA fragment was 200-500 bp). Cell lysate was centrifuged (20,000×g, 20 min, 4 °C) and supernatant was collected and precleared by protein-G Plus agarose (Santa Cruz sc2002) in RIPA buffer. 2 mg of total proteins were mixed with 2 µg of anti-HA high affinity antibody (Roche, cat. no.: 11867423001) and incubated at 4 °C for 16 hours. Sample was washed three times with RIPA buffer, four times with WASH buffer (Triton X-100, 0.5 %; Sodium deoxycholate, 0.4 %; LiCl, 0.5 M; Tris, pH 8.5, 100 mM), two more times with RIPA buffer and twice with TE buffer (EDTA, 10 mM; Tris, pH 8, 10 mM) followed by elution with Elution buffer (SDS, 1%; EDTA,10 mM; Tris, pH 8, 50 mM). Finally, the sample was decrosslinked by overnight incubation with 200 mM NaCl and Proteinase K at 60 °C overnight. DNA was purified by NucleoSpin gDNA Clean-up (Macherey-Nagel). ChIP-seq libraries were prepared by TaKaRa ThruPLEX DNA-Seq kit from input amounts of DNA ranging from 50 to 1000 pg. Depending on DNA input library amplification cycles varied from 11 to 16. Individual libraries were quantified by Qubit fluorometer High Sensitivity DNA Assay and fragment profiles were analyzed by Agilent 2100 Bioanalyzer High Sensitivity DNA Assay