title: GSM1937971: Fur IP ChIP-Seq Anaerobic, Iron Deficient B; Escherichia coli str. K-12 substr. MG1655; ChIP-Seq
library_construction_protocol: Cell pellets (from initial 250 mL of culture) were thawed and resuspended in 250 μL of IP buffer (100 mM Tris pH 8, 300 mM NaCl, 1% TritonX-100) and sonicated using a microtip sonicator set at 10% output for 20 second intervals with periods of cooling in between. Cells were then treated for one hour at 4 °C with RNase A (2 ng/ml), micrococcal nuclease (50 units), 20 μM CaCl2,1.2 mM KCl, 0.3 mM NaCl, 6 mM sucrose, and 10 μM DTT. EDTA was added to 10 mM to stop the micrococcal nuclease and the samples were spun down to remove cell debris. The lysate was then precleared through incubation with a 50/50 slurry of sepharose protein A beads in IP buffer for 2-3 hours at 4 °C. The beads were removed by centrifugation and antibody was added to the pre-cleared lysate for an overnight incubation. The next day, 30 μl of a 50/50 slurry of sepharose protein A beads in IP buffer was added to the lysate to capture antibody-protein-DNA complex for one hour at 4 °C. Beads were then washed once with 1 ml of LiCl wash buffer (100 mM Tris pH 8, 250 mM LiCl, 2% TritonX-100), twice with 600 mM NaCl wash buffer (100 mM Tris pH 8, 600 mM NaCl, 2% TritonX-100), twice with 300 mM NaCl wash buffer (100 mM Tris pH 8, 300 mM NaCl, 2% TritonX-100), and twice with TE. Elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) was added after the final wash step, and beads were incubated at 65 °C for 30 minutes to remove the crosslinked protein-DNA complexes from the beads. After centrifugation to remove the beads, the samples were incubated overnight at 65 °C to reverse the protein-DNA formaldehyde crosslinks. DNA was purified using Qiagen’s PCR Purification kit and eluted to a final volume of 50 μl with EB. DNA samples were submitted to the University of Wisconsin-Madison DNA Sequencing Facility for ChIP-seq library preparation. All libraries were generated using reagents from the Illumina Paired End Sample Preparation Kit (Illumina) and the Illumina protocol “Preparing Samples for ChIP Sequencing of DNA” (Illumina part # 11257047 RevA) as per the manufacturer’s instructions, except products of the ligation reaction were purified by gel electrophoresis using 2% SizeSelect agarose gels (Invitrogen) targeting 50 bp fragments.  After library construction and amplification, quality and quantity were assessed using an Agilent DNA 1000 series chip assay (Agilent) and QuantIT PicoGreen dsDNA Kit (Invitrogen), respectively, and libraries were standardized to 10μM.  Cluster generation was performed using a cBot Single Read Cluster Generation Kit (v4) and placed on the Illumina cBot.  For aerobic and anaerobic replicates A, a single read, 50 bp run was performed, using standard SBS kits (v4) and SCS 2.6 on an Illumina Genome Analyzer IIx. For aerobic and anaerobic replicates B and C, the input, and the anaerobic, iron deficient samples, a single read, 50 bp run was performed, using standard SBS kits (v3) and SCS 1.8.2 on an Illumina HiSeq2000.
identifiers.namespace: GEO
identifiers.id: GSM1937971