title: GSM1847009: pstS_PYE_control; Caulobacter vibrioides NA1000; ChIP-Seq
library_construction_protocol: Cross-linked DNA was extracted as in Fioravanti et al., 2013 (PMID: 23737758) with modifications: mid-exponential phase cultures were cross-linked in 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde at room temperature for 10 minutes. Reactions were quenched with 0.1 M glycine at room temperature for 5 minutes, and on ice for 15 minutes. Cells were washed 3X in phosphate buffered saline (PBS) and lysed with Ready-Lyse lysozyme solution (Epicentre, Madison, WI) according to manufacturer instructions. Lysates were diluted 1:1 in ChIP buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl) plus Roche Protease Inhibitor Tablets (Roche) and incubated at 37 °C for 10 minutes. Lysates were sonicated (Branson Sonicator) on ice for 6 bursts of 10 seconds each at 15% amplitude, and then cleared by centrifugation at 14,000 rpm for 5 minutes at 4 °C. Cleared supernatants were normalized by protein content in 1 mL of ChIP buffer + 0.01% SDS, and pre-cleared with 50 μL of Protein-A DynaBeads (Invitrogen) (pre-blocked with 100 μg Ultra Pure BSA in ChIP buffer + 0.01% SDS) by 1 hour rotation at 4 °C.  10% of each supernatant was removed and used as total chromatin input sample. The remaining supernatant was incubated with a 1:1000 dilution of anti-M2 antibody overnight at 4 °C. Each sample was then incubated with 50 μL of pre-blocked Protein-A DynaBeads for 6 hours at 4 °C with rotation. DynaBeads were washed consecutively at 4 °C for 15 minutes with 1 mL of the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)), and twice with TE buffer (10 mM Tris-HCl (pH 8.1), 1 mM EDTA). Complexes were eluted twice by incubation with 250 μL freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) at 30 °C for 15 minutes.  Cross-links were reversed by addition of 300 mM NaCl and 2 μL of 0.5 mg/mL RNase A and overnight incubation at 65 °C. Samples were treated with 5 μL of Proteinase K (20 mg/mL, NEB) in 40 mM EDTA and 40 mM Tris-HCl (pH 6.8) for 2 hours at 45 °C. DNA was extracted using a PCR purification kit (Qiagen) and resuspended in 80 μL of water.  Libraries were prepared using the SPRI-works system and sequenced on an Illumina MiSeq (MIT BioMicroCenter). Libraries were prepared using the SPRI-works system
attributes.tag: GEO Accession
attributes.value: GSM1847009