title: GSM3359900: Fur Deficient Iron #2; Bacillus subtilis; ChIP-Seq
library_construction_protocol: The cell pellets were washed and resuspended with buffer CA (10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 137 mM NaCl, and 2.7 mM KCl). The samples were incubated with 1% formaldehyde at room temperature for 10 min for crosslinking and then incubated with 133 mM glycine (pH7.5) at 4 °C for 30 min to quench the crosslinking. Cells were spun down, washed twice with buffer CB (50 mM Tris–HCl pH7.4, 150 mM NaCl and 1 mM EDTA), and then resuspended in 0.5 ml buffer CB followed by sonication for cell lysis and DNA fragmentation. Supernatant was collected after centrifugation and the total protein concentration was quantified using a Bradford assay. Aliquots of 400 µg of total protein were kept at −80 °C. Aliquots of 1% volume of the lysate were diluted with CB buffer and kept at −80 °C to serve as the input-control (1% of input DNA). For immunoprecipitation, α-FLAG M2 magnetic agarose beads (Sigma, Cat# M8823) were washed and resuspended in 400 μl buffer CB. Aliquots of 400 µg of total protein were diluted, mixed with the washed magnetic beads, and incubated on a rotation mixer overnight in a cold room (40C). The bead slurry was recovered by using a magnetic stand and washed twice with 500 μl of buffer CB. The protein–DNA complexes were eluted with 3X FLAG peptide according to the manufacture's protocol. All samples including 1% input DNA samples were treated at 650C for overnight to reverse crosslinking. Co-immunoprecipitated DNA was purified using a PCR purification Kit (Omega Biotek, Norcross, GA), quantified by NanoDrop spectrophotometer. Fifty to one hundred nanograms of ChIP or input DNA was used for DNA library preparation at the Cornell University Life Sciences Core Laboratories Center.