|
| Status |
Public on Mar 22, 2016 |
| Title |
exp_NCL_2 |
| Sample type |
genomic |
| |
|
| Source name |
Bacterial cells
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
treatment: Psoralen + UV
genotype: Wild-type
growth phase: Mid-Exponential
|
| Treatment protocol |
Cells from 25 mL culture were harvested by centrifugation at the given time points and washed with cold phosphate buffer (pH7.2). The cells were resuspended in 10 ml Tris-Cl buffer (pH 8) and incubated at 37oC for 2 minutes. EDTA (final 0.5 mM) was added and cells were incubated at the same temperature for another two minutes. The cells were immediately chilled on ice and MgCl2 (final 1 mM) was added. 100 μl of saturated trimethylpsoralen solution in ethanol (Sigma-Aldrich, cat.no T6137) was added to the chilled cell suspension, mixed by gentle shaking and incubated at 4oC for 10 minutes. The cells were then exposed to UV light of wavelength 365 nM and intensity 1.2 kJ.m-2min-1 for 45 seconds, immediately washed with M69 buffer and finally resuspended in the same buffer.
|
| Growth protocol |
Cells were grown in LB medium at 37 C with aeration, until mid-exponential phase (OD600=0.95) or stationary phase (OD600=2.4).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by adding EDTA (final 0.1 M) and 2% SDS and incubating at 37 C for 1 hour followed by addition of 100 μl proteinase K and overnight incubation at the same temperature. After cell lysis, genomic DNA was isolated by the phenol-chloroform method, treated overnight with DNase free Rnase and purified by the phenol-chloroform method. DNA was resuspended in tris buffer (pH 8) and sheared by sonication to a median length of 300 bp. Sheared DNA was electrophoresced in an agarose gel, eluted, concentrated by ethanol precipitation and dissolved in 25 mm sodium phosphate buffer (pH 7). The DNA was heated at 95 oC for 2 minutes followed by quick addition of DMSO (final 50%) and glyoxal (final 6-8 % by volume) and reheating at 60 oC for two hours. Glyoxal treated DNA fragments were separated on a 3.5 % agarose gel in phosphate buffer. The gel was incubated with denaturing solution (0.5 M NaOH, 1.5 M NaCl) at 65 oC for 3 hours to reverse psoralen crosslinks. Crosslinked and non-crosslinked DNA fragments were excised separately from the gel, eluted, and concentrated by ethanol precipitation.
|
| Label |
biotin
|
| Label protocol |
Fragmented DNA was 3’ termini biotin labeled using the GeneChip® DNA Labeling Reagent (Affymetrix 900542) and 60U of Terminal Deoxynucleotidyl Transferase (Promega M1875) at 37°C for 60 minutes. The labeling reaction was stopped by the addition of 0.5M EDTA.
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| |
|
| Hybridization protocol |
The crosslinked and non-crosslinked DNA was hybridized separately to high resolution tiling microarrays. Labeled DNA fragments (3ug) were hybridized for 16 hours (60 rpms) at 45°C to tiling array chips (Ecoli_Tab520346F, GPL8585) purchased from Affymetrix
|
| Scan protocol |
Hybridized, washed and stained microarrays were scanned using a Genechip Scanner 3000 (Affymetrix) according to manufacturer's instructions.
|
| Description |
exp.2.txt
Non-crosslinked fraction
|
| Data processing |
Probe intensities were corrected for background by subtracting the median intensities of control (non-E. coli) probes having the same G+C content. This was followed by quantile normalization of signals from all arrays and log2 transformation. For each probe, log-transformed signals from the non-crosslinked DNA samples were subtracted from those of the corresponding crosslinked DNA samples. The resultant values of log2(crosslinked signal/non-crosslinked signal) were taken as a measure of psoralen binding to each sequence. These were smoothed using a moving average with a sliding window of size 1100 bp.
txt files with 3 columns. Column 1: probe sequence. Column 2: start position of the probe on the E. coli genome. Column 3: log2(crosslinked signal/non-crosslinked signal)
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| |
|
| Submission date |
Feb 08, 2016 |
| Last update date |
Mar 23, 2016 |
| Contact name |
Avantika Lal |
| E-mail(s) |
avantika@ncbs.res.in
|
| Organization name |
National Centre for Biological Sciences
|
| Street address |
GKVK, Bellary Road
|
| City |
Bangalore |
| ZIP/Postal code |
560065 |
| Country |
India |
| |
|
| Platform ID |
GPL8585 |
| Series (1) |
| GSE77687 |
Psoralen binding across the Escherichia coli chromosome |
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