GSE54669-GSM1321544-GPL8708-PMID:24606919.tsv 6.51 KB
"genotype: MG1655"	"characteristics_ch1.1"
"treatment: None"	"characteristics_ch1.2"
"antibody: anti-DksA rabbit polyclonal antisera"	"characteristics_ch1.3"
"genotype: MG1655"	"characteristics_ch2.1"
"treatment: None"	"characteristics_ch2.2"
"antibody: None, input DNA"	"characteristics_ch2.3"
"The log2-ratio is computed and scaled to center the ratio data around zero. Scaling is performed by subtracting the bi-weight mean for the log2-ratio values for all features on the array from each log2-ratio value."	"data_processing.1"
"Cells were fixed with 1% formaldehyde for 5min before quenching with ice-cold glycine (100mM). Cells were harvested, washed with ice-cold PBS and flash-frozen in liquid nitrogen. Cell pellets were resuspended in 500 ml of IP buffer (100 mM Tris pH 8, 300mM NaCl, 2% TritonX-100) and sonicated using a Misonix sonicator (S-4000) with a cup horn (431C) set at 60% output, 10 sec ON and 10 sec OFF, for a total sonication time of 16 min. Cells were then treated for one hour at 4 °C with RNase A (2 ng/ml; USB, Inc.) and the samples were centrifuged at 20,000 x g for 10 min at 4 °C to remove cell debris. The lysate was then incubated with a 50/50 slurry of Sepharose protein A beads (Upstate; now Millipore) and protein G beads (GE Healthcare) in IP buffer for 3 hours at 4 °C. The beads were removed by centrifugation (1000 x g for 2 min at 4 °C) and antibodies were added to the pre-cleared lysate for an overnight incubation. For ChIP, we used anti-RNAP b subunit NT63 monoclonal antibodies (Neoclone W0002), anti-RNAP s70 subunit monoclonal antibodies (Neoclone W0004), or anti-DksA rabbit polyclonal antisera (a kind gift from Diana Downs). The next day, 30 μl of a 50/50 slurry of Sepharose protein A and G beads in 250 μl 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 250 mM LiCl wash buffer (100 mM Tris pH 8, 250 mM LiCl, 2% Triton X-100), twice with 1 ml 600 mM NaCl wash buffer (100 mM Tris pH 8, 600 mM NaCl, 2% SDS), twice with 1 ml 300 mM NaCl wash buffer (100 mM Tris pH 8, 300 mM NaCl, 2% SDS), and twice with 1 ml TE (10 mM Tris pH 8, 1 mM EDTA). 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 (1000 x g for 2 min at 25 °C) 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 in a final volume of 65 μl with 10 mM Tris pH 8."	"extract_protocol_ch1.1"
"Cells were fixed with 1% formaldehyde for 5min before quenching with ice-cold glycine (100mM). Cells were harvested, washed with ice-cold PBS and flash-frozen in liquid nitrogen. Cell pellets were resuspended in 500 ml of IP buffer (100 mM Tris pH 8, 300mM NaCl, 2% TritonX-100) and sonicated using a Misonix sonicator (S-4000) with a cup horn (431C) set at 60% output, 10 sec ON and 10 sec OFF, for a total sonication time of 16 min. Cells were then treated for one hour at 4 °C with RNase A (2 ng/ml; USB, Inc.) and the samples were centrifuged at 20,000 x g for 10 min at 4 °C to remove cell debris. The lysate was then incubated with a 50/50 slurry of Sepharose protein A beads (Upstate; now Millipore) and protein G beads (GE Healthcare) in IP buffer for 3 hours at 4 °C. The beads were removed by centrifugation (1000 x g for 2 min at 4 °C) and antibodies were added to the pre-cleared lysate for an overnight incubation. For ChIP, we used anti-RNAP b subunit NT63 monoclonal antibodies (Neoclone W0002), anti-RNAP s70 subunit monoclonal antibodies (Neoclone W0004), or anti-DksA rabbit polyclonal antisera (a kind gift from Diana Downs). The next day, 30 μl of a 50/50 slurry of Sepharose protein A and G beads in 250 μl 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 250 mM LiCl wash buffer (100 mM Tris pH 8, 250 mM LiCl, 2% Triton X-100), twice with 1 ml 600 mM NaCl wash buffer (100 mM Tris pH 8, 600 mM NaCl, 2% SDS), twice with 1 ml 300 mM NaCl wash buffer (100 mM Tris pH 8, 300 mM NaCl, 2% SDS), and twice with 1 ml TE (10 mM Tris pH 8, 1 mM EDTA). 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 (1000 x g for 2 min at 25 °C) 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 in a final volume of 65 μl with 10 mM Tris pH 8."	"extract_protocol_ch2.1"
"Wild-types cells were grown in MOPS medium with 0.2% glucose, leucine, isoleucine, valine, glycine, phenylalanine, threonine (40 mg/ml) and uracil (50 mg/ml) until mid-log phase (OD600~0.4) at 37°C with vigorous shaking."	"growth_protocol_ch1.1"
"Wild-types cells were grown in MOPS medium with 0.2% glucose, leucine, isoleucine, valine, glycine, phenylalanine, threonine (40 mg/ml) and uracil (50 mg/ml) until mid-log phase at 37°C with vigorous shaking."	"growth_protocol_ch2.1"
"Escherichia coli"	"organism_ch1.1"
"Escherichia coli"	"organism_ch2.1"
"DksA ChIP DNA from wt cells"	"source_name_ch1.1"
"Input DNA from wt cells"	"source_name_ch2.1"
"DksA_wt_untreated_rep2"	"title.1"
"None"	"treatment_protocol_ch1.1"
"None"	"treatment_protocol_ch2.1"
"genotype: MG1655"	"characteristics_ch1.1"
"treatment: None"	"characteristics_ch1.2"
"antibody: anti-DksA rabbit polyclonal antisera"	"characteristics_ch1.3"
"genotype: MG1655"	"characteristics_ch2.1"
"treatment: None"	"characteristics_ch2.2"
"antibody: None, input DNA"	"characteristics_ch2.3"
"Wild-types cells were grown in MOPS medium with 0.2% glucose, leucine, isoleucine, valine, glycine, phenylalanine, threonine (40 mg/ml) and uracil (50 mg/ml) until mid-log phase (OD600~0.4) at 37°C with vigorous shaking."	"growth_protocol_ch1.1"
"Wild-types cells were grown in MOPS medium with 0.2% glucose, leucine, isoleucine, valine, glycine, phenylalanine, threonine (40 mg/ml) and uracil (50 mg/ml) until mid-log phase at 37°C with vigorous shaking."	"growth_protocol_ch2.1"
"Escherichia coli"	"organism_ch1.1"
"Escherichia coli"	"organism_ch2.1"
"DksA ChIP DNA from wt cells"	"source_name_ch1.1"
"Input DNA from wt cells"	"source_name_ch2.1"
"None"	"treatment_protocol_ch1.1"
"None"	"treatment_protocol_ch2.1"