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Sample GSM412596

Query DataSets for GSM412596

Status

Public on Sep 01, 2009

Title

genomic_ref5

Sample type

genomic

Source name

MG1655 genomic DNA

Organism

Escherichia coli

Characteristics

strain: MG1655

Treatment protocol

For cross-linking, 30 mL of cells were mixed with 300 uL 1M sodium phosphates (pH 7.6) and 810 uL 37% formaldehyde. Cross-linking was quenched by addition of 2 mL 2M glycine. Protein-DNA complexes were isolated from cells grown to early (2.4 x 10^7 CFU/ml) or late (2.4 x 10^8 CFU/ml) exponential phase. Batch cultures of E. coli MDS42 were grown to early exponential phase. To obtain tiling-resolution RNA measures across the E. coli genome, wild-type cells were grown in biological duplicate in LB to early exponential phase. The cultures were moved to ice, and the Qiagen RNeasy kit (P/N 74104) was used to isolate total cellular RNA.

Growth protocol

Batch cultures of E. coli MG1655 were grown at 37°C with shaking in Luria-Bertani medium (0.1% Bacto Tryptone, 0.05% yeast extract, 0.05% NaCl).

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA Extraction: Genomic DNA was extracted according to Current Protocols in Molecular Biology. Protein-DNA complex isolation: Protein-DNA complexes were isolated by phenol extraction with 150 μL 10 mM Tris and 500 μL 25:24:1 phenol : chloroform : isoamyl alcohol. A white disk was readily discernible at the aqueous/organic interface. To purify this interface, all aqueous and organic liquid was removed by pipetting. A second extraction was performed by adding 500 μL 10 mM Tris and 450 μL 24:1 chloroform: isoamyl alcohol, vortexing and centrifuging. Again, all liquid was removed from the interface by pipetting, and residual liquid was removed. For cross-link reversal: the interface was suspended in 500 μL 10 mM Tris and 50 μL 10% SDS, and placed at ~100 ˚C for 30 minutes. The tubes were placed on ice, then moved to 65 ˚C for 3 hours following addition of 5 μL proteinase K (20 mg/mL). After heat treatment, the solutions were phenol/chloroform extracted and ethanol precipitated in the presence of glycogen to purify the DNA. RNA extraction: the Qiagen RNeasy kit (P/N 74104) was used to isolate total cellular RNA. Immediately following elution in RNase-free water, residual DNA was removed by treatment with DNaseI at 37 °C for 15 minutes. The samples were treated again using the RNeasy kit, resuspended in 40 μl RNase-free water, and stored at -20 °C.

Label

Biotin

Label protocol

DNA labeling: Two ug of the DNA isolated from DNA-protein complexes was used in a labeling reaction with the Enzo Terminal Labeling kit (P/N 42630). The labeling reactions were assembled according to the manufacturer’s instructions; the labeling reaction was incubated at 37 ˚C for 30 minutes to 1 hour. RNA expression profiling: RNA samples were reverse-transcribed to DNA using the SuperScript system from Invitrogen (P/N 18053017). Ten ug of RNA was incubated with 5 ug random hexamer primer at 70 °C for 10 minutes, then mixed with 8 uL SuperScript buffer, 4 uL DTT, 2 uL 10 uM dNTP mix, 3 uL DEPC-treated water, 1 uL RNasin and 2 uL of SuperScript II Enzyme. The samples was incubated at 25 °C for 10 minutes, and 42 °C for 2.5 hours, then moved to 95 °C for 5 minutes to terminate the reaction. To fragment the RNA template, 2 uL 1N NaOH was added, and the samples were placed at 65 °C for 15 minutes. At room temperature, the pH was readjusted by adding 2 uL 1N HCl. The cDNA was cleaned using the Qiagen QiaQuick Nucleotide Removal Kit (P/N 28304).

Hybridization protocol

The FS450_0002 washing protocol (Affymetrix) was used. Briefly, this includes two post-hybridization washes, a streptavidin-phycoerythrin stain, a post-stain wash, an antibody amplification stain, a second streptavidin-phycoerythrin stain, a final wash, and addition of holding buffer to the microarray.

Scan protocol

After completion of the wash cycle, the microarrays were scanned a single time at room temperature on a Affymetrix GeneChip Scanner 3000.

Description

genomic_ref5
Each raw data file represents a biological replicate.

Data processing

We developed in-house computational and statistical analysis tools for use with the E. coli tiling array. We used a previous study (Choe et al., 2005) as a model for perfect match adjustment on single arrays. Analysis scripts were written in Perl, MatLab, and R to standardize the statistical manipulations across all data sets. The output file from the scanning process is a CEL file. The raw CEL data were used as the basis for the manuscript; no processed data are available.

Submission date

Jun 03, 2009

Last update date

Sep 01, 2009

Contact name

yirchung Liu

E-mail(s)

yliu@princeton.edu

Organization name

Princeton University

Department

Molecular Biology