SL1344 cells were inoculated 1:100 into 25 ml fresh Luria Broth (LB) and grown at 37degrees celsius in a shaking waterbath until OD600 2.0
Extracted molecule
genomic DNA
Extraction protocol
25 ml of culture were harvested and re-suspended in 50 ml of PBS. DNA-protein interactions were cross-linked by adding formaldehyde to a final concentration of 1% for 30 minutes. Glycine was then added to a final concentration of 0.125M to stop the cross-linking. The cells were pelleted, re-suspended in 0.6 ml of lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, Roche protease inhibitor cocktail). 1.4 ml of IP dilution buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, Roche protease inhibitor cocktail) was added and the chromatin was sonicated on ice in a 5ml tube to reduce the DNA length to an average size of approximately 500 bp using the Sanyo/MES Soniprep sonicator (8 bursts at an amplitude 10 microns for 30 seconds, with 1 minute cooling between bursts). 1.0 ml of IP dilution buffer was added to the chromatin, which was then pre-cleared by adding 50 ul of normal rabbit IgG for 1 hour at 4°C on a rotating wheel. 100 ul of homogeneous Protein G-agarose suspension was added to the pre-cleared chromatin and the samples were incubated for 3 hours at 4°C on a rotating wheel. The samples were centrifuged at 7500 rpm for 2 minutes at 4°C to pellet the protein G-agarose beads and the supernatant was used to set up various immunoprecipitation conditions in 2 ml microfuge tubes. An aliquot of 200 ul of chromatin was stored at -20°C to be used as input DNA. Experimental ChIP conditions – 1350 ul chromatin + 10 μl of mouse monoclonal to the RNAP Polymerase β' subunit (Neoclone, catalogue number W0001) or 10 ug of mouse monoclonal to RpoD (Neoclone, catalogue number W0004). The samples were incubated at 4°C overnight on a rotating wheel. 50 μl of homogeneous protein G-agarose suspension was added to each sample and the samples were incubated at 4°C for at least 3 hours on a rotating wheel. The samples were centrifuged at 7,500 rpm for 2 minutes at 4°C to pellet the protein G-agarose beads. The supernatant was removed and the protein G-agarose beads were washed twice with 750 ul of cold IP wash buffer 1 (20 mM Tris-HCl pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with 750 ul of cold IP wash buffer 2 (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid) and twice with 750 ul of cold TE pH 8.0. DNA-protein-antibody complexes were eluted from the protein G-agarose beads by twice washing the beads with 225 ul of IP elution buffer (100 mM NaHCO3, 1% SDS) and both eluates were combined. 0.2 ul of RNase A (10 mg/ml) and NaCl (to a final concentration of 0.3 M) were added to each of the IP test and input samples and incubated at 65ºC for 6 hours to reverse the cross-links. 9 ul of Proteinase K (10 mg/ml) was then added to each sample and incubated at 45ºC for at least 3 hours. DNA was purified and recovered by performing a standard phenol-chloroform extraction, followed by ethanol precipitation with 5 ug of glycogen and 5 ug of yeast tRNA. The DNA pellets of the IP samples were re-suspended in 50 ul of sterile filtered water and 100 ul for the Input DNA samples
Label
Cy3
Label protocol
Fluorescent labeling of DNA samples were carried out using the BioPrime Random Labeling kit (Invitrogen) as follows: 60 ul of 2.5 x random primer solution, x* ul DNA, and (70.5-x) ul sterile water were heated at 100oC for 10 minutes, snap chilled on ice and following were then added – 15 ul dNTP mix (1mM dCTP, 2mM dGTP, dTTP, dATP), 1.5 ul 1 mM Cy3/Cy5 labeled dCTP (GE healthcare) and 3 ul Klenow fragment (40 U/ ul). *[20 ul of ChIP DNA and 300 ng of Input DNA were fluorescently labeled with Cy3 d-CTP and Cy5-dCTP respectively]. The Labeling reactions wereas carried out at 37 oC overnight and 15 ml of stop buffer added to terminate the reaction. Labeled DNAs were purified using G-50 columns (GE-healthcare), according to manufacturers instructions.
SL1344 cells were inoculated 1:100 into 25 ml fresh Luria Broth (LB) and grown at 37degrees celsius in a shaking waterbath until OD600 2.0
Extracted molecule
genomic DNA
Extraction protocol
25 ml of culture were harvested and re-suspended in 50 ml of PBS. DNA-protein interactions were cross-linked by adding formaldehyde to a final concentration of 1% for 30 minutes. Glycine was then added to a final concentration of 0.125M to stop the cross-linking. The cells were pelleted, re-suspended in 0.6 ml of lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, Roche protease inhibitor cocktail). 1.4 ml of IP dilution buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, Roche protease inhibitor cocktail) was added and the chromatin was sonicated on ice in a 5ml tube to reduce the DNA length to an average size of approximately 500 bp using the Sanyo/MES Soniprep sonicator (8 bursts at an amplitude 10 microns for 30 seconds, with 1 minute cooling between bursts). 1.0 ml of IP dilution buffer was added to the chromatin, which was then pre-cleared by adding 50 ul of normal rabbit IgG for 1 hour at 4°C on a rotating wheel. 100 ul of homogeneous Protein G-agarose suspension was added to the pre-cleared chromatin and the samples were incubated for 3 hours at 4°C on a rotating wheel. The samples were centrifuged at 7500 rpm for 2 minutes at 4°C to pellet the protein G-agarose beads and the supernatant was used to set up various immunoprecipitation conditions in 2 ml microfuge tubes. An aliquot of 200 ul of chromatin was stored at -20°C to be used as input DNA. Experimental ChIP conditions – 1350 ul chromatin + 10 μl of mouse monoclonal to the RNAP Polymerase β' subunit (Neoclone, catalogue number W0001) or 10 ug of mouse monoclonal to RpoD (Neoclone, catalogue number W0004). The samples were incubated at 4°C overnight on a rotating wheel. 50 μl of homogeneous protein G-agarose suspension was added to each sample and the samples were incubated at 4°C for at least 3 hours on a rotating wheel. The samples were centrifuged at 7,500 rpm for 2 minutes at 4°C to pellet the protein G-agarose beads. The supernatant was removed and the protein G-agarose beads were washed twice with 750 ul of cold IP wash buffer 1 (20 mM Tris-HCl pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with 750 ul of cold IP wash buffer 2 (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid) and twice with 750 ul of cold TE pH 8.0. DNA-protein-antibody complexes were eluted from the protein G-agarose beads by twice washing the beads with 225 ul of IP elution buffer (100 mM NaHCO3, 1% SDS) and both eluates were combined. 0.2 ul of RNase A (10 mg/ml) and NaCl (to a final concentration of 0.3 M) were added to each of the IP test and input samples and incubated at 65ºC for 6 hours to reverse the cross-links. 9 ul of Proteinase K (10 mg/ml) was then added to each sample and incubated at 45ºC for at least 3 hours. DNA was purified and recovered by performing a standard phenol-chloroform extraction, followed by ethanol precipitation with 5 ug of glycogen and 5 ug of yeast tRNA. The DNA pellets of the IP samples were re-suspended in 50 ul of sterile filtered water and 100 ul for the Input DNA samples
Label
Cy5
Label protocol
Fluorescent labeling of DNA samples were carried out using the BioPrime Random Labeling kit (Invitrogen) as follows: 60 ul of 2.5 x random primer solution, x* ul DNA, and (70.5-x) ul sterile water were heated at 100oC for 10 minutes, snap chilled on ice and following were then added – 15 ul dNTP mix (1mM dCTP, 2mM dGTP, dTTP, dATP), 1.5 ul 1 mM Cy3/Cy5 labeled dCTP (GE healthcare) and 3 ul Klenow fragment (40 U/ ul). *[20 ul of ChIP DNA and 300 ng of Input DNA were fluorescently labeled with Cy3 d-CTP and Cy5-dCTP respectively]. The Labeling reactions wereas carried out at 37 oC overnight and 15 ml of stop buffer added to terminate the reaction. Labeled DNAs were purified using G-50 columns (GE-healthcare), according to manufacturers instructions.
Hybridization protocol
Cy3 labeled ChIP DNA and Cy5 labeled control genomic DNAs were co-precipitated using standard sodium acetate/ethanol procedures and resuspended in hybridization buffer (Oxford Gene Technologies), and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers and hybridiized for 24 hours at 55oC in an Agilent hybiridization oven at 5 rpm. After hybridization, slides were washed according to instructions provided by Oxford Gene Technologies
Scan protocol
The microarray slides were scanned using an Agilent G2505C scanner. Cy3 and Cy5 images were acquired at 3 micron resolution. Scanned images were analyzed using Feature extraction software (Agilent).
Description
biological replicate 2
Data processing
median normalization of background subtracted Cy3/Cy5 (532/635) ratio values
