GSM5050089: Gbn_ChIP_25h; Streptomyces coelicolor; ChIP-Seq
After 25 h or 48 h growth, cellophane disks were soaked up-side-down in PBS solution containing 1% formaldehyde for 20 min allowing DNA-protein crosslink. Ten plates were collected for 25 h samples; four plates were collected for 48 h samples. Then the disks were moved to PBS solution containing 0.5 M glycine for 5 min to stop crosslinking reaction. The mycelium was then collected, washed in PBS and resuspended in 0.5 mL lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 15 mg·mL-1 lysozyme, 1× protease inhibitor (Roche, Bavaria, 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% v/v Triton-X-100) was added to the sample, and chromosomal DNA sheared to 100 to 500 bp using Bioruptor Plus water bath sonication system (Diagenode, Liège, Belgium). After centrifugation to remove cell debris, lysates were incubated with 40 uL Anti-FLAG M2 affinity gel (cat A2220, Sigma-Aldrich, St. Louis, U.S.) suspension according to the manufacturer's instructions and incubated at 4°C overnight. After centrifugation and washing, the pellets and 50 uL of untreated total extracts (controls) were incubated in 100 uL IP elution buffer (50 mM Tris-HCl pH7.5, 10 mM EDTA, 1% m/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 DNA Clean & Concentrator kit (Cat. D4029, Zymo Research, California, U.S.). The libraries were prepared by BGI for sequencing using BGISEQ-500 platform. Briefly, the fragmented DNA was end-repaired and 3'-dA overhang was added to the product. Methylated sequencing adaptors were then ligated and the library was PCR amplified and size selected (100-300bp, including adaptor sequence). DNA-end repair, 3'-dA overhang and ligation of methylated sequencing adaptors. PCR amplification and size selection (100-300bp, including adaptor sequence).