strain: SL1344 chip antibody: Fis growth condition: grown for 2 hours under non-aerated conditions
Treatment protocol
Subsequently, cells were fixed, lysed and sonicated. At this point, sheared chromatin (Ch) DNA from all culture samples was immunoprecipitated (IP), in which case it was either added IgG or FLAG-tag antibody, and both named IP test samples. DNA carrying FLAG-tag antibody was named ChIP Flagged DNA, and DNA carrying IgG was named ChIP IgG DNA. The DNA without either antibody, but subsequently frozen at -80ºC, was used later for the chip microarray portion as an internal DNA control, which was labeled input or mock IP DNA. In order to determine the Fis enrichment in either ChIP DNA samples, real-time quantitative PCR was done in their respective extracted DNA to measure the relative levels of promoter and gene regions of known Fis-binding sites. ChIP Flagged DNA was compared to ChIP IgG DNA, which was used as a control.
Growth protocol
Constructed strain SG07 fis::3XFlag was used for this set of experiments to identify peaks of Fis-binding on gene regions that are occupied by Fis. Bacterial cells were grown in Luria Bertani medium, under non-aerated conditions, and harvested from mid-log (2 hr) phase. All culture samples were normalized at a total OD A600 of 10, as culture samples replicates from each growth phase and condition were combined to result in that total OD.
Extracted molecule
genomic DNA
Extraction protocol
Immunoprecipitated DNA Chromatin (ChIP DNA) and input or mock immunoprecipitated (IP) DNA from culture samples were extracted using phenol (Sigma) and chloroform, and concentrated by ethanol precipitation. A volume of 2 μl of yeast tRNA (5 mg/ml, Invitrogen) was added to each sample (except input IP) just before adding 250 μl of phenol (Sigma) and 250 μl of chloroform. Samples were vortexed and centrifuged at 13000 rpm for 5 min at room temperature. The aqueous layer (top layer) was carefully removed and placed in a fresh tube. A volume of 500 μl of chloroform was added to each sample. The samples were vortexed and centrifuged at 13000 rpm for 5 min at room temperature. The aqueous layer was again transferred to a fresh microfuge tube. At this point, 5 μg of glycogen (5 mg/ml, Roche), 1 μl of yeast tRNA (5 mg/ml, Invitrogen) (except input IP), and 50 μl (20 μl for input IP) of 3M NaAc (pH 5.2) was added to each sample and mixed well. The DNA was precipitated with 1375 μl (550 μl for input IP) of 100% ethanol and incubated at -70ºC from 30 min to 1 to 2 hours maximum (or -20°C overnight). The samples were centrifuged at 13000 rpm for 20 min at 4ºC. The DNA pellets were washed with 500 μl of ice-cold 70% ethanol and air-dried for 10-15 minutes. The DNA pellets of the IP samples were resuspended in 50 μl of sterile distilled water and 100 μl for the Input DNA samples. Samples were stored at -20°C.
Label
Cy3
Label protocol
Labelling by random priming of DNA samples: The DNA was labelled using BioPrime Random Labelling Kit (Invitrogen) as described below. Labelling method used for array hybridization as follows: reagents, 2.5 X Random primer solution; ChIP Flagged IP DNA or Input DNA; and sterile H2O were mixed on ice in a microfuge tube. The DNA amount labelled was different for Input and Flag-tagged IP DNA samples. DNA quantity labelled was 40 μl of unamplified Flag-tagged IP DNA and approximately 200 ng of Input DNA. This mixture was heated at 100°C for 10 min to denature the DNA and then chilled briefly on ice. The following reagents were added to the tubes on ice: 10 X dNTP mix, 1 mM Cy3/Cy5 labelled dCTP (1 mM Cy3-dCTP, 1 mM Cy5-dCTP, GE Healthcare). Input IP mock DNA samples were labelled with Cy5 (blue) dCTP, and ChIP Flagged IP DNA samples were labelled with Cy3 (red) dCTP) and 40 U/μl of Klenow fragment. The final volume per labelling reaction was 150 μl. The reagents were mixed gently but thoroughly and incubated at 37°C overnight. Stop buffer (10 mM EDTA) was added to the reaction mix to terminate the reaction. Purification of labelled DNA samples: Labelled DNA samples were purified as follows. Micro-spin G50 columns (GE Healthcare) were used to remove the unlabelled nucleotides from the labelled DNA samples. Three columns were used for each of the labelling reactions. The resin was resuspended in the columns by vortexing gently. The caps were loosened and the bottom of the tubes snapped off. The columns were placed in 2 ml microfuge tubes and centrifuged at 4000 rpm for 1 min. A volume of 50 μl of sterile filtered HPLC water was applied to the resin-bed and the columns were centrifuged at 4000 rpm for 1 min. The columns were placed in fresh 1 ml microfuge tubes and the labelled DNA samples were carefully applied to the resin-bed. The columns were then centrifuged at 13000 rpm for 1 min. The purified DNA samples were collected in the 1.5 ml microfuge tubes and the samples (Input + ChIP) from the same labelled reaction were pooled together. The final volume for the labelled DNA samples was approximately 180 μl. A volume of 5 μl of each labelled DNA was analyzed on a 1% agarose 1 X TBE gel and stained with ethidium bromide for visualization of smeared DNA. One tenth of the sample volume of 3M sodium acetate was added to precipitate DNA. Two and a half volumes of 100% ETOH were added to each sample. Samples were subsequently incubated at –80ºC in the dark for 1-3 hrs or, at –20ºC overnight. DNA was pelleted after spinning in the dark for 20 min at RT and at 13000 rpm. Alcohol was drained and pellet resuspended in 80% ETOH. The tubes were allowed to air-dry for 15-30 min in a dark place. Pellet was resuspended in 100 μl Hybridization buffer (1M Sodium chloride, 50 mM MES (Sodium salt SIGMA) (pH 6.5), 20 mM EDTA, 20% formamide (SIGMA), and 1% Triton X100). Labelled DNA was subsequently heated at 70ºC for 10 min and denatured at 100ºC for another 10 min. It was briefly placed on ice prior to hybridization.
strain: SL1344 chip antibody: None, Input DNA growth condition: grown for 2 hours under non-aerated conditions
Treatment protocol
Subsequently, cells were fixed, lysed and sonicated. At this point, sheared chromatin (Ch) DNA from all culture samples was immunoprecipitated (IP), in which case it was either added IgG or FLAG-tag antibody, and both named IP test samples. DNA carrying FLAG-tag antibody was named ChIP Flagged DNA, and DNA carrying IgG was named ChIP IgG DNA. The DNA without either antibody, but subsequently frozen at -80ºC, was used later for the chip microarray portion as an internal DNA control, which was labeled input or mock IP DNA. In order to determine the Fis enrichment in either ChIP DNA samples, real-time quantitative PCR was done in their respective extracted DNA to measure the relative levels of promoter and gene regions of known Fis-binding sites. ChIP Flagged DNA was compared to ChIP IgG DNA, which was used as a control.
Growth protocol
grown for 2 hours under non-aerated conditions
Extracted molecule
genomic DNA
Extraction protocol
Immunoprecipitated DNA Chromatin (ChIP DNA) and input or mock immunoprecipitated (IP) DNA from culture samples were extracted using phenol (Sigma) and chloroform, and concentrated by ethanol precipitation. A volume of 2 μl of yeast tRNA (5 mg/ml, Invitrogen) was added to each sample (except input IP) just before adding 250 μl of phenol (Sigma) and 250 μl of chloroform. Samples were vortexed and centrifuged at 13000 rpm for 5 min at room temperature. The aqueous layer (top layer) was carefully removed and placed in a fresh tube. A volume of 500 μl of chloroform was added to each sample. The samples were vortexed and centrifuged at 13000 rpm for 5 min at room temperature. The aqueous layer was again transferred to a fresh microfuge tube. At this point, 5 μg of glycogen (5 mg/ml, Roche), 1 μl of yeast tRNA (5 mg/ml, Invitrogen) (except input IP), and 50 μl (20 μl for input IP) of 3M NaAc (pH 5.2) was added to each sample and mixed well. The DNA was precipitated with 1375 μl (550 μl for input IP) of 100% ethanol and incubated at -70ºC from 30 min to 1 to 2 hours maximum (or -20°C overnight). The samples were centrifuged at 13000 rpm for 20 min at 4ºC. The DNA pellets were washed with 500 μl of ice-cold 70% ethanol and air-dried for 10-15 minutes. The DNA pellets of the IP samples were resuspended in 50 μl of sterile distilled water and 100 μl for the Input DNA samples. Samples were stored at -20°C.
Label
Cy5
Label protocol
Labelling by random priming of DNA samples: The DNA was labelled using BioPrime Random Labelling Kit (Invitrogen) as described below. Labelling method used for array hybridization as follows: reagents, 2.5 X Random primer solution; ChIP Flagged IP DNA or Input DNA; and sterile H2O were mixed on ice in a microfuge tube. The DNA amount labelled was different for Input and Flag-tagged IP DNA samples. DNA quantity labelled was 40 μl of unamplified Flag-tagged IP DNA and approximately 200 ng of Input DNA. This mixture was heated at 100°C for 10 min to denature the DNA and then chilled briefly on ice. The following reagents were added to the tubes on ice: 10 X dNTP mix, 1 mM Cy3/Cy5 labelled dCTP (1 mM Cy3-dCTP, 1 mM Cy5-dCTP, GE Healthcare). Input IP mock DNA samples were labelled with Cy5 (blue) dCTP, and ChIP Flagged IP DNA samples were labelled with Cy3 (red) dCTP) and 40 U/μl of Klenow fragment. The final volume per labelling reaction was 150 μl. The reagents were mixed gently but thoroughly and incubated at 37°C overnight. Stop buffer (10 mM EDTA) was added to the reaction mix to terminate the reaction. Purification of labelled DNA samples: Labelled DNA samples were purified as follows. Micro-spin G50 columns (GE Healthcare) were used to remove the unlabelled nucleotides from the labelled DNA samples. Three columns were used for each of the labelling reactions. The resin was resuspended in the columns by vortexing gently. The caps were loosened and the bottom of the tubes snapped off. The columns were placed in 2 ml microfuge tubes and centrifuged at 4000 rpm for 1 min. A volume of 50 μl of sterile filtered HPLC water was applied to the resin-bed and the columns were centrifuged at 4000 rpm for 1 min. The columns were placed in fresh 1 ml microfuge tubes and the labelled DNA samples were carefully applied to the resin-bed. The columns were then centrifuged at 13000 rpm for 1 min. The purified DNA samples were collected in the 1.5 ml microfuge tubes and the samples (Input + ChIP) from the same labelled reaction were pooled together. The final volume for the labelled DNA samples was approximately 180 μl. A volume of 5 μl of each labelled DNA was analyzed on a 1% agarose 1 X TBE gel and stained with ethidium bromide for visualization of smeared DNA. One tenth of the sample volume of 3M sodium acetate was added to precipitate DNA. Two and a half volumes of 100% ETOH were added to each sample. Samples were subsequently incubated at –80ºC in the dark for 1-3 hrs or, at –20ºC overnight. DNA was pelleted after spinning in the dark for 20 min at RT and at 13000 rpm. Alcohol was drained and pellet resuspended in 80% ETOH. The tubes were allowed to air-dry for 15-30 min in a dark place. Pellet was resuspended in 100 μl Hybridization buffer (1M Sodium chloride, 50 mM MES (Sodium salt SIGMA) (pH 6.5), 20 mM EDTA, 20% formamide (SIGMA), and 1% Triton X100). Labelled DNA was subsequently heated at 70ºC for 10 min and denatured at 100ºC for another 10 min. It was briefly placed on ice prior to hybridization.
Hybridization protocol
Hybridizations The entire volume of each labelled DNA sample was carefully pipetted over each of the four gaskets in a glass slide (Oxford Gene Technology, Agilent). The order of samples from top to bottom, were as follows: aerated mid-log, aerated late-stationary phase, non-aerated mid-log, and non-aerated late-stationary phase. For orientation reference, the slide barcode was always at bottom. Subsequently, the microarray slide (Oxford Gene Technology, Agilent) was placed carefully on top of the loaded gaskets slide, avoiding any bubble formation and with its reference barcode in the same orientation. Both slides were kept against each other for adequate hybridization in a tight metallic holder. Slides were incubated in the hybridization oven (Shel Lab, Hybridizer) at 55ºC overnight (24 hrs).
Scan protocol
Genome-wide chip microarray scanning The hybridized microarray slide was separated from the gasket slide and washed twice in buffer 1 (20X SSPE (SIGMA), 10% Tween and sterile ddH2O), and twice again in buffer 2 (20X SSPE (SIGMA), 100% Polyethylene Glycol (PEG) (Fluka)). The microarray slide was blot dried from the corners with a regular paper towel prior to scan reading. The slide was inserted into a microarray scanner (Axon) array side up (barcode side facing down). In order to view the microarray, each oligonucleotide on an OGT services array is assigned a unique feature number. The identity of the feature number is given by a CD-ROM with GenePix Array List (GAL) files.
Description
Biological replicate 2 of 3.
Data processing
From the scan output the F532/F635 Median values were selected for foreground intensities and B532/B635 Median values for background intensities. Background was subtracted from foreground. Any spot with background-corrected intensity below 1 was discharded. Spots that were flagged manually ('Flag' column), automatically ('Autoflag' column) or considered a control spot ('ControlType' column) were also skipped. For the remaining spots the log2-ratios (log2(test/control)) were calculated. The final values for the matrix were generated by subtracting the median log2-ratio of each slide from the log2-ratios of each spot on that slide. These values were then read into ChiPOTle (Buck et al., 2005) for peak detection.
