6 protocols
AccessionNameType
P-AFFY-6
scanning
Title: Affymetrix CEL analysis. Description:
P-MTAB-30695
hybridization
According to the manufacturer's instruction for Prokayotic Target Hybridization by Affymetrix degrees (Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 4, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 200, Volume unit = Micro litre, temperature = 45)
P-MTAB-30694
labeling
PCR-amplified DNA fragments were digested by DNase I (GE healthcare Bioscience) treatment. The digested DNA fragments were terminally labeled with biotin-ddUTP using ENZO BioArray Terminal Labeling Kit (ENZO life sciences) degrees (Parameters: Amplification = none, Mass unit = Micro gram)
P-MTAB-30692
grow
W3110 hns-3xflag Km (TON1816) and W3110 hha::km ydgT::cm hns-3xflag (TU01) cells were grown in 200 ml of LB (+0.3 M NaCl) medium under aerobic conditions at 37 degrees C until the culture reached an OD600 of 0.4. degrees (Parameters: time unit = seconds, min temperature = 37, temperature unit = C)
P-MTAB-30693
nucleic_acid_extraction
The cultures were treated with formaldehyde (final concentration, 1%) for 30 min at room temperature to crosslink H-NS-Flag to the chromosomal DNA. The excess formaldehyde was quenched by incubation for 10 min with 1.5 ml of 3M glycine solution. Cells were harvested, washed with Tris-buffered saline (TBS), and lysed with lysis buffer (10 mM Tris-HCl pH 8.0, 20% sucrose, 50 mM NaCl, and 10 mM EDTA). Washed cells were suspended in 500 ul of lysis buffer containing 20 mg/ml lysozyme and incubated for 30 min at 37 degrees C. The samples were then treated with 4 ml of IP buffer (50 mM HEPES-KOH pH 7.5, 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 5% glycerol) and phenylmethylsulfonyl fluoride (PMSF; final concentration, 1 mg/ml). The cells were sonicated (XL2020; Astrason, USA) on ice for 1 min, repeated 10 times at 1 min intervals. The disrupted cell solutions were clarified by centrifugation at 15,000 rpm for 30 min at 4 degrees C. A part of supernatant fraction (whole cell extract) in each sample was recovered and proteins in whole cell extracts were digested with 2 mg/ml proteinase K (Takara, Japan) at 42 degrees C for 2 hr, followed by incubation at 65 degrees C for 6 hr to decrosslink H-NS-Flag and the chromosomal DNA, and to inactivate the proteinase K. Free DNA fragments in the whole cell extracts (sup DNA) were purified with a QIAquick purification kit (Qiagen, Germany) and eluted with 100 ul of the provided elution buffer. The recovered DNA was PCR amplified as described previously (see reference). Reference Katou, Y., Kaneshiro, K., Aburatani, H. and Shirahige, K. 2006, Genomic approach for the understanding of dynamic aspect of chromosome behavior, Methods Enzymol., 409, 389-410. degrees (Parameters: Extracted product = genomic_DNA, Amplification = none)
P-MTAB-30696
nucleic_acid_extraction
The cultures were treated with formaldehyde (final concentration, 1%) for 30 min at room temperature to crosslink H-NS-Flag to the chromosomal DNA. The excess formaldehyde was quenched by incubation for 10 min with 1.5 ml of 3M glycine solution. Cells were harvested, washed with Tris-buffered saline (TBS), and lysed with lysis buffer (10 mM Tris-HCl pH 8.0, 20% sucrose, 50 mM NaCl, and 10 mM EDTA). Washed cells were suspended in 500 ul of lysis buffer containing 20 mg/ml lysozyme and incubated for 30 min at 37 degrees C. The samples were then treated with 4 ml of IP buffer (50 mM HEPES-KOH pH 7.5, 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 5% glycerol) and phenylmethylsulfonyl fluoride (PMSF; final concentration, 1 mg/ml). The cells were sonicated (XL2020; Astrason, USA) on ice for 1 min, repeated 10 times at 1 min intervals. The disrupted cell solutions were clarified by centrifugation at 15,000 rpm for 30 min at 4 degrees C. The supernatant (whole cell extract) was mixed with anti-FLAG antibody (Sigma-Aldrich, Germany)-coated-protein A Dynal Dynabeads (100.02; Invitrogen, Norway), which were prepared as previously described (see reference). The beads were incubated at 4 degrees C overnight with rotation, and then rinsed twice with IP buffer for 10 min with rotation at 4 degrees C, once with IP salt buffer (IP buffer containing 500 mM NaCl), once with wash buffer (10mM Tris-HCl pH8.0, 250mM LiCl, 1mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate), and then once with TE (10 mM Tris-HCl pH8.0, 1 mM EDTA pH 8.0). The Flag-tagged H-NS-bound DNA fragments were released from the beads by addition of 100 ul elution buffer (250 mM Tris-HCl pH 7.5, 50 mM EDTA pH8.0, 5% SDS) followed by heating at 65 degrees C for 20 min. Flag-tagged H-NS in immunoprecipitated DNA fractions were digested with 2 mg/ml proteinase K (Takara, Japan) at 42 degrees C for 2 hr, followed by incubation at 65 degrees C for 6 hr to decrosslink H-NS-Flag and the chromosomal DNA, and to inactivate the proteinase K. Free DNA fragments in the immunoprecipitated DNA fractions (ChIP DNA) were purified with a QIAquick purification kit (Qiagen, Germany) and eluted with 100 ul of the provided elution buffer. The recovered DNA was PCR amplified as described previously (see reference). Reference Katou, Y., Kaneshiro, K., Aburatani, H. and Shirahige, K. 2006, Genomic approach for the understanding of dynamic aspect of chromosome behavior, Methods Enzymol., 409, 389-410. degrees (Parameters: Extracted product = genomic_DNA, Amplification = none)