title: GSM7468676: mannitol and GlcN, 24h, ChIP sample; Streptomyces coelicolor A3(2); ChIP-Seq
library_construction_protocol: The mycelium was collected, washed in PBS, and resuspended in 0.5 mL lysis buffer (10 mM Tris-HCl [pH 8.0], 50 mM NaCl, 15 mg  mL21 lysozyme, 1 protease inhibitor; Roche, Germany) and incubated at 37°C for 20 min. After incubation, 0.5 mL IP buffer (100 mM Tris-HCl [pH 8.0], 250 mM NaCl, 0.8% [vol/vol] Triton X-100) was added to the sample, and chro- mosomal DNA was sheared to 100 to 500 bp using the Bioruptor Plus water bath sonication system (Diagenode, Liège, Belgium). After centrifugation to remove cell debris, lysates were incubated with 40 mL anti-FLAG M2 affinity gel (catalog no. A2220; Sigma-Aldrich, St. Louis, MO) suspension according to the manufacturer's instructions and incubated at 4°C overnight. After centrifugation and washing, the pellets and 50 mL of untreated total extracts (controls) were incubated in 100 mL IP elution buffer (50 mM Tris-HCl [pH 7.5], 10 mM EDTA, 1% [w/v] SDS) at 65°C overnight to reverse cross-links. Beads were then removed by centrifugation before DNA extraction with phenol-chloroform. The DNA sample was then extracted with chloroform, and the water layer was further purified using a DNA Clean & Concentrator kit (catalog no. D4029; Zymo Research, CA). The construction of the libraries is through the processes of end repairing, adding A to tails, purification, PCR amplification and so on.