title: GSM1417297: FnrL_ChIP_Seq_Rep2; Rhodobacter sphaeroides; ChIP-Seq
library_construction_protocol: At midexponential phase (~2×108 colony-forming units/ml), formaldehyde and sodium phosphate were added to a final concentration of 1% and 10 mM, respectively. This mixture was incubated at 30 °C for 4 min before glycine was added to 100 mM, and the solution was placed on ice for 30 min with gentle agitation to quench excess formaldehyde. Cells were centrifuged at 3000g, washed twice with chilled phosphate buffered saline, centrifuged, and flash-frozen at −80 °C. About 2×1010 cells were suspended in 0.5 ml of 100 mM Tris (pH 8.0), 300 mM NaCl, 2% Triton X-100, RNase A (0.5 μg) and 1 mM phenylmethylsulfonyl fluoride, and sonicated in a Misonix water bath for 8 mins with 10s pulses and intervals. Cell debris was removed by centrifugation for 10 min at 12,000g, and an aliquot was removed to analyze DNA fragmentation by agarose gel eletrophoresis (desired size of ~200–1000 bp with enrichment for ~500-bp molecules). The resulting supernatant was incubated with gentle mixing with 20 μl of Staphylococcus aureus protein A Sepharose beads (Sigma-Aldrich, St. Louis, MO) for 3 h at 4 °C as pretreatment to remove potential nonspecific binding to the beads. After the beads had been removed by centrifugation (5 min at 3000g), one-tenth of the sample was removed and used as non-antibody-treated control. Five microlitre of Myc epitope antibody (ab9132, Abcam plc) or FnrL polyclonal antibody was added and the mixture was incubated overnight at 4 °C with gentle mixing before being incubated with protein A Sepharose beads (30 μl) for 3 h at 4 °C. The beads were recovered by centrifugation; washed once (4 °C) with 250 mM LiCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; washed twice with 600 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; washed twice with 300 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; and washed twice with TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA). Protein–DNA complexes were eluted from the beads by incubating at 65 °C for 30 min in 50 mM Tris (pH 8.0), 10 nM EDTA, and 1% sodium dodecyl sulfate. The beads were removed by centrifugation, and protein–DNA cross-linking was reversed by incubating the samples for 12 h at 65 °C. DNA was purified using the QIAquick PCR Purification Kit. Samples were sent to UW-Madison Biotech center for further processing and sequencing. Libraries were prepared according to Illumina's instructions by UW-Madison biotech center
attributes.tag: GEO Accession
attributes.value: GSM1417297