Fresh mycelia were incubated in 200mM CaCl2 solution for 1 hour with shaking.
Growth protocol
Fungal mycelia were grown in CM for 2 days.
Extracted molecule
genomic DNA
Extraction protocol
Mycelia were cross-linked with 1% formaldehyde in buffer A (0.4M sucrose, 10mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% formaldehyde) for cross-linking for 20min. Mycelia were harvested with excess amount of distilled water after stopping cross-link with 0.1M glycine for 10 min, frozen in liquid nitrogen, ground into a fine powder in a chilled mortar and pestle, and stored at -80C until used. Nuclear DNA was then isolated from cross-linked mycelia with Plant Nuclear Isolation Kit (Sigma, St. Louis, MO) and sheared into fragments by sonication to 200- to 1,000-bp with an average size of 500bp. Sonication was conducted on ice with an amplitude of 10% using 30× 30s pulses (30s between bursts) using Biorupter (Cosmo Bio, Tokyo, Japan). After pre-clearing nuclear lysates with Salmon sperm/protein A agarose (Upstate, Temecula, CA) for 4 hours at 4C, immunoprecipitations were performed with either 1 µg of rabbit control IgG (ab46540-1, Abcam, Cambridge, MA) or 0.5µl of antiGFP antibody (ab290, Abcam) for overnight at 4C. A small aliquot of DNA (30%) was saved for input DNA (input). Immunoprecipitated DNA was captured with proteinA agarose beads (Upstate, Temecula, CA) for 4 hours, and then washed twice with LNDET buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA) and twice with TE buffer. The DNAs were reverse-cross linked at 65C overnight in elution buffer (1% SDS and 0.1 M NaHCO3) containing 1 mg/ml proteinase K, and purified using PCR purification kit (Qiagen).
Label
Cy5
Label protocol
DNA was then labeled with Cy3 or Cy5 fluorescent dyes for input or immunoprecipitated DNA, respectively, according to the NimbleGen's protocol.
Fresh mycelia were incubated in 200mM CaCl2 solution for 1 hour with shaking.
Growth protocol
Fungal mycelia were grown in CM for 2 days.
Extracted molecule
genomic DNA
Extraction protocol
Mycelia were cross-linked with 1% formaldehyde in buffer A (0.4M sucrose, 10mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% formaldehyde) for cross-linking for 20min. Mycelia were harvested with excess amount of distilled water after stopping cross-link with 0.1M glycine for 10 min, frozen in liquid nitrogen, ground into a fine powder in a chilled mortar and pestle, and stored at -80C until used. Nuclear DNA was then isolated from cross-linked mycelia with Plant Nuclear Isolation Kit (Sigma, St. Louis, MO) and sheared into fragments by sonication to 200- to 1,000-bp with an average size of 500bp. Sonication was conducted on ice with an amplitude of 10% using 30× 30s pulses (30s between bursts) using Biorupter (Cosmo Bio, Tokyo, Japan). After pre-clearing nuclear lysates with Salmon sperm/protein A agarose (Upstate, Temecula, CA) for 4 hours at 4C, immunoprecipitations were performed with either 1 µg of rabbit control IgG (ab46540-1, Abcam, Cambridge, MA) or 0.5µl of antiGFP antibody (ab290, Abcam) for overnight at 4C. A small aliquot of DNA (30%) was saved for input DNA (input). Immunoprecipitated DNA was captured with proteinA agarose beads (Upstate, Temecula, CA) for 4 hours, and then washed twice with LNDET buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA) and twice with TE buffer. The DNAs were reverse-cross linked at 65C overnight in elution buffer (1% SDS and 0.1 M NaHCO3) containing 1 mg/ml proteinase K, and purified using PCR purification kit (Qiagen).
Label
Cy3
Label protocol
DNA was then labeled with Cy3 or Cy5 fluorescent dyes for input or immunoprecipitated DNA, respectively, according to the NimbleGen's protocol.
Hybridization protocol
Labeled DNA was hybridized according to NimbleGen's protocol.
