|
| Status |
Public on Jun 14, 2011 |
| Title |
PurR_Adenine_2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
E. Coli PurR ChIP DNA Adenine 2
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
growth condition: Adenine
genotype: PurR-8myc
chip antibody: 9E10 Myc tag antibody
|
| Treatment protocol |
Cross-linked and sonicated chromatin complex of PurR-8myc and DNA was immunoprecipitated by 9E10 myc antibody.
|
| Growth protocol |
E. coli strains harboring PurR-8myc were grown in minimal M9 medium supplemented with glucose (2 g/L) then inoculated into 100mL of fresh M9 minimal medium supplemented with 100ug/L adenine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells at appropriate cell density were cross-linked by 1% formaldehyde at room temperature for 25 min. Following quenching the unused formaldehyde with a final concentration of 125 mM glycine at room temperature for 5 min. The cross-linked cells were harvested and washed three times with 50 mL of ice-cold TBS (Tris Buffered Saline). The washed cells were re-suspended in 0.5 mL lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 ug/mL RNaseA, protease inhibitor cocktail (Sigma) and 1 kU Ready-LyseTM lysozyme (Epicentre). The cells were incubated at room temperature for 30 min and then treated with 0.5 mL of 2XIP buffer with the protease inhibitor cocktail. The lysate was then sonicated four times for 20 sec each in an ice bath to fragment the chromatin complexes using Misonix sonicator 3000 (output level = 2.5). The range of the DNA size resulting from the sonication procedure was 300 – 1000 bp. 6 uL of mouse antibody (NT63, Neoclone) was used to immunoprecipitate the chromatin complex of RNA polymerase subunit (rpoB) and DNA. For the control (mock-IP), 2 ug of normal mouse IgG (Upstate) was added into the cell extract. IP DNAs were purified with QIAquick PCR Purification Kit (Qiagen) then amplified PCR.
|
| Label |
Cy5
|
| Label protocol |
Performed by Nimblegen following their standard operating protocol (www.nimblegen.com)
|
| |
|
| Channel 2 |
| Source name |
E. Coli PurR Input DNA Adenine 2
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
growth condition: Adenine
genotype: PurR-8myc
antibody: normal mouse IgG (Upstate)
|
| Treatment protocol |
Cross-linked and sonicated chromatin complex of PurR-8myc and DNA was immunoprecipitated by using normal mouse IgG for the control.
|
| Growth protocol |
E. coli strains harboring PurR-8myc were grown in minimal M9 medium supplemented with glucose (2 g/L) then inoculated into 100mL of fresh M9 minimal medium supplemented with 100ug/L adenine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells at appropriate cell density were cross-linked by 1% formaldehyde at room temperature for 25 min. Following quenching the unused formaldehyde with a final concentration of 125 mM glycine at room temperature for 5 min. The cross-linked cells were harvested and washed three times with 50 mL of ice-cold TBS (Tris Buffered Saline). The washed cells were re-suspended in 0.5 mL lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 ug/mL RNaseA, protease inhibitor cocktail (Sigma) and 1 kU Ready-LyseTM lysozyme (Epicentre). The cells were incubated at room temperature for 30 min and then treated with 0.5 mL of 2XIP buffer with the protease inhibitor cocktail. The lysate was then sonicated four times for 20 sec each in an ice bath to fragment the chromatin complexes using Misonix sonicator 3000 (output level = 2.5). The range of the DNA size resulting from the sonication procedure was 300 – 1000 bp. 6 uL of mouse antibody (NT63, Neoclone) was used to immunoprecipitate the chromatin complex of RNA polymerase subunit (rpoB) and DNA. For the control (mock-IP), 2 ug of normal mouse IgG (Upstate) was added into the cell extract. IP DNAs were purified with QIAquick PCR Purification Kit (Qiagen) then amplified PCR.
|
| Label |
Cy3
|
| Label protocol |
Performed by Nimblegen following their standard operating protocol (www.nimblegen.com)
|
| |
|
| |
| Hybridization protocol |
Performed by Nimblegen following their standard operating protocol (www.nimblegen.com)
|
| Scan protocol |
Performed by Nimblegen following their standard operating protocol (www.nimblegen.com)
|
| Description |
E. coli strains harboring PurR-8myc chromatin immunoprecipitation under adenine conditions.
|
| Data processing |
The raw data (.pair file) was subjected to per channel quantile normalization (Bolstad et al. Bioinformatics 19(2):185), IP/mock-IP ratio computation and enriched region identification as implemented in the NimbleScan software package, version 2.4.27 (www.nimblegen.com).
|
| |
|
| Submission date |
Jan 12, 2011 |
| Last update date |
Jun 14, 2011 |
| Contact name |
Bernhard Palsson |
| E-mail(s) |
palsson@ucsd.edu
|
| Phone |
858-534-5668
|
| Fax |
858-822-3120
|
| URL |
http://systemsbiology.ucsd.edu
|
| Organization name |
UCSD
|
| Department |
Bioengineering
|
| Lab |
Systems Biology Research Group
|
| Street address |
9500 Gilman Dr.
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92122 |
| Country |
USA |
| |
|
| Platform ID |
GPL8387 |
| Series (2) |
| GSE26589 |
ChIP-chip of E. coli K-12 MG1655 with antibody against PurR-8myc under various conditions. |
| GSE26591 |
Genome-scale reconstruction of the PurR regulon reveals its role in the adenine stimulon of Escherichia coli K-12 MG1655 |
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