|
| Status |
Public on Feb 18, 2015 |
| Title |
RosR_ChIP_noH2O2_rep6_dyeswap |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
0258_21_swap
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: NRC-1 transformed with VNG0258H(RosR)::myc
ip: myc epitope
condition: no H2O2 (non-stress)
|
| Treatment protocol |
Cultures were either left untreated or exposed to 25 mM H2O2 for 10, 20, and 60 minutes as indicated in Sample Names
|
| Growth protocol |
Halobacterium salinarum NRC-1 harboring VNG0258H(RosR) fused at its C-terminus to the myc epitope was grown to mid-logarithmic phase (OD600 ~ 0.2 - 0.4) in complete medium (CM; 250 g/L NaCl, 20 g/L MgSO4·7H2O, 3 g/L sodium citrate, 2 g/L KCl, 10 g/L peptone) containing 20ug/mL mevinolin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Transcription factor-chromatin complexes from each treated time point and untreated cultures were then cross-linked in vivo with 1% formaldehyde for 30 min at room temperature and subjected to immunoprecipitation (IP) by virtue of the myc epitope tag as described previously (Schmid et al. 2011).
|
| Label |
cy5
|
| Label protocol |
DNA fragments were directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously (Facciotti et al., 2007). Briefly, one µg of each IP sample was labeled with Cy3 or Cy5 dyes from Kreatch corporation according to manufacturer instructions. Matched, mock-treated controls were oppositely labeled. Dye swaps were conducted to correct for bias in incorporation.
|
| |
|
| Channel 2 |
| Source name |
control
|
| Organism |
Halobacterium salinarum NRC-1 |
| Characteristics |
strain: NRC-1 transformed with VNG0258H(RosR)::myc
ip: mock
condition: no H2O2 (non-stress)
|
| Treatment protocol |
Cultures were either left untreated or exposed to 25 mM H2O2 for 10, 20, and 60 minutes as indicated in Sample Names
|
| Growth protocol |
Halobacterium salinarum NRC-1 harboring VNG0258H(RosR) fused at its C-terminus to the myc epitope was grown to mid-logarithmic phase (OD600 ~ 0.2 - 0.4) in complete medium (CM; 250 g/L NaCl, 20 g/L MgSO4·7H2O, 3 g/L sodium citrate, 2 g/L KCl, 10 g/L peptone) containing 20ug/mL mevinolin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Transcription factor-chromatin complexes from each treated time point and untreated cultures were then cross-linked in vivo with 1% formaldehyde for 30 min at room temperature and subjected to immunoprecipitation (IP) by virtue of the myc epitope tag as described previously (Schmid et al. 2011).
|
| Label |
cy3
|
| Label protocol |
DNA fragments were directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously (Facciotti et al., 2007). Briefly, one µg of each IP sample was labeled with Cy3 or Cy5 dyes from Kreatch corporation according to manufacturer instructions. Matched, mock-treated controls were oppositely labeled. Dye swaps were conducted to correct for bias in incorporation.
|
| |
|
| |
| Hybridization protocol |
Labeled samples were hybridized to custom 2 x 105,000 feature 60-mer oligonucleotide microarray (Agilent Technologies, AMADID 026819). On this high-resolution array, the entire H. salinarum genome was tiled every 30 bp in triplicate. Randomly selected regions of the genome were spotted in quadruplicate. Microarray slide hybridization and washing protocols were conducted according to the manufacturer’s instructions (Agilent technologies) with the exception that hybridization was conducted in the presence of 37.5% formamide at 68˚C to ensure proper stringency.
|
| Scan protocol |
Slides were scanned on an Agilent G2566AAl scanner using the ChIP-v1_95_May07 protocol.
|
| Data processing |
Agilent Feature Extraction was used to map raw spot intensities to microarray grid. Raw probe intensities were normalized first within each array using density-weighted loess (Lickwar et al., 2012). Second, probes were normalized to quantiles across arrays. Binding peaks were detected from normalized data for each replicate independently using MeDiChI in R (Reiss et al., 2008).
|
| |
|
| Submission date |
Jun 20, 2014 |
| Last update date |
Feb 18, 2015 |
| Contact name |
Amy K Schmid |
| E-mail(s) |
amy.schmid@duke.edu
|
| Organization name |
Duke University
|
| Department |
Biology
|
| Lab |
Schmid
|
| Street address |
125 Science Dr.
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27707 |
| Country |
USA |
| |
|
| Platform ID |
GPL18848 |
| Series (1) |
| GSE58696 |
A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea. |
|
