GSM2231315: Mutant 10h; Streptomyces venezuelae ATCC 10712; ChIP-Seq
A spore inoculum (~5-10 ul of 1x10^8 spores) sufficient to reach an OD600 of 0.35 after 8 hours of growth was added to 35ml of MYM tap media in 250 ml flasks containing springs.  Following growth to the chosen time point, the entire content of the flask was transferred to a 50 ml falcon tube for crosslinking, which was carried out by incubation at 30oC for 30 mins with 1% final concentration of formaldehyde (v/v).  Crosslinking was quenched by incubation at room temperature with glycine (final concentration of 125 mM).  Mycelium was harvested by centrifugation 4000 rpm at 4oC for 10 min and washed twice with ice cold PBS before transfer to a 2 ml centrifuge tube.  Pellets were resuspended in 0.75 ml lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mg/ml lysozyme, 1x protease inhibitor-Roche1186170001) and incubate at 37C for 10-25 min.  Then 0.75 ml 1x IP buffer (100mM tris-HCl pH 8.0, 250 mM NaCl, 0.5% Triton x-100, 0.1% SDS, 1x protease inhibitor (Roche)) was added and samples mixed by pipetting up and down.  Samples were sonicated 7x at 50Hz, 10 sec/cycle with a 1 min incubation on ice after each cycle.  DNA fragmentation was checked by agarose gel electrophoresis following phenol extraction of 25 µl of the crude lysate mixed with 75 µl of TE buffer with 100-200 µl of phenol/chloroform.   Contaminating RNA was removed with 2 µl RNase (1mg/ml) added to extracted DNA followed by an incubation for 30 min at 37°C. A smear of DNA from 200 to 1000 bp with the majority of DNA 200-400 bp should be visible. Crude lysate was centrifuged at 13,000 rpm for 15 minutes at 4°C to clear the sample of cell debris.  M2 affinity beads (Sigma-Aldrich #A2220) were prepared by washing in ½IP buffer following manufacturers instructions.  The cleared lysate was incubated with 40 µl of washed M2 beads and incubated for 4 h at 4C in a vertical rotor.  The lysate was removed and the beads pooled into one 1.5 microfuge tube and washed in 0.5 IP buffer.  The beads were transferred to a fresh microfuge. tube and washed a further 3 times removing as much buffer as possible without disturbing the beads.  The DNA-protein complex was eluted from the beads with 100 µl elution buffer (50 mM Tris-HCl pH7.6, 10mM EDTA, 1% SDS) by incubating at 65°C overnight.  Removing the ~100µl elution buffer, an extra 50 µl of elution buffer was added and further incubated at 65oC for 5 min.   To extract the DNA 150 µl eluate, 2 µl proteinase K (10 mg/ml) was added and incubated 1.5 hrs at 55oC.   To the reaction 150 µl phenol-chloroform was added.  Samples were vortexed and centrifuged at full speed for 10 min.  The aqueous layer was extracted and purified using the Qiaquick column from Qiagen with a final elution using 50 µl EB buffer (Qiagen).   The concentration of samples were determined using Quant-iT™ PicoGreen ® dsDNA Reagent (Invitrogen) or equivalent kit or by nanodrop measurement. Cells were treated with formaldehyde to crosslink DNA and protein, lysed, sonicated and the tagged protein/DNA complexes were immunoprecipitated using anti-flag antibody bound M2 beads. DNA libraries were prepared for sequencing using standard Illumina protocols.
GEO Accession
GSM2231315