GSM7047597: S. coelicolor FLAG_CutR, no glucose, rep 2; Streptomyces coelicolor; ChIP-Seq
To cross-link proteins to DNA the cellophane disks were removed and submersed in 10 ml fresh 1% (v/v) formaldehyde solution at room temperature for 20 minutes. After a 5-minute incubation in 10 ml 0.5 M glycine the mycelium was harvested and washed twice with 25 ml ice-cold PBS pH 7.4 before being flash frozen in liquid nitrogen and stored at -80°C. For lysis the pellets were resuspended in 2 ml lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mg/ml lysozyme, EDTA-free protease inhibitor) before 750 μl was transferred to a 2 ml microcentrifuge and samples incubated at 37°C for 30minutes. For fragmentation 750 μl IP buffer (100 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.5% v/v Triton X-100, 0.1% SDS, EDTA-free protease inhibitor) was added and samples sonicated on ice at 50 Hz for 20 cycles of 10 seconds with at least 1 minute of rest between each cycle. 25 μl of the crude lysate was combined with 75 μl TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and extracted with 100 μl phenol:chloroform. 2 μl 1mg/ml RNase A was mixed with 25 μl of the resulting extract, incubated at 37°C for 30 minutes and run on a 1% agarose gel to confirm a smear centred at 500 bp. The remaining crude lysate was centrifuged for 15 minutes at 13000 rpm, 4°C and supernatant saved. To prepare for binding 750 μl of Anti-FLAG M2 magnetic beads (Sigma Aldrich) were washed in 3.75 ml 0.5 IP buffer. The bead slurry was then mixed with 40 μl clarified lysate and incubated overnight on a vertical rotator  The lysate was removed, and the beads washed 4 times with 500 μl 0.5 IP buffer including 10 minutes vertical rotation at 4°C each cycle. Washed beads were incubated in 100 μl elution buffer (50 mM Tris-HCl pH 7.6, 10 mM EDTA, 1% SDS) overnight at 65°C. Elutant was removed and saved before an additional 50 μl elution  buffer was added and incubated at 65°C for a further 5 minutes. Once removed 3 μl 10mg/ml proteinase K was added to the combined eluants and incubated at 55°C for 2 hours. DNA was extracted with 200 μl phenol:chloroform and purified using a QIAquick PCR Purification Kit (QIAGEN) following the manufacturer's instructions. DNA was eluted in 50 μl Buffer EB (10 mM Tris-HCl pH 8.5) and 47 μl snap frozen in liquid nitrogen and stored at -80°C. The remaining 3 μl of sample was used to determine the DNA concentration and quality. The Qubit Fluorometer 2.0 with the high-sensitivity kit was used for DNA concentration whilst DNA purity was quantified using a Nanodrop™ 2000 UV-Vis Spectrophotometer. Carried out by Novogene