|
| Status |
Public on Jun 30, 2017 |
| Title |
FrvA induced vs uninduced_60 min |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
IPTG induced
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype: Pspac-frvA
background strain: CU1065
|
| Treatment protocol |
To induce expression of FrvA, 1 mM IPTG was added into one flask while the other was left untreated as a control.
|
| Growth protocol |
Cells (WT Pspac-frvA) were grown in LB medium amended with 10 µM to an OD600 of ∼0.25 and divided into two 1 L flasks, one treated with 1 mM IPTG and the other untreated. Aliquots of 40 ml of cell culture were harvested from both flasks at different time-points.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using an acidic phenol-based method.
|
| Label |
Alexa Fluor 555
|
| Label protocol |
Total RNA were then treated with Turbo-DNAse (Thermo Fisher Scientific) to remove any possible DNA contamination, and then reverse transcribed using SuperScript™ Indirect cDNA Labeling System (Thermo Fisher Scientific) into cDNA. cDNA was aliquoted and labelled with either fluorescent dye 555 or dye 647, and hybridized to microarray slides containing 60 nt oligonucleotides for each coding region according to laboratory protocol.
|
| |
|
| Channel 2 |
| Source name |
uninduced
|
| Organism |
Bacillus subtilis |
| Characteristics |
genotype: Pspac-frvA
background strain: CU1065
|
| Treatment protocol |
To induce expression of FrvA, 1 mM IPTG was added into one flask while the other was left untreated as a control.
|
| Growth protocol |
Cells (WT Pspac-frvA) were grown in LB medium amended with 10 µM to an OD600 of ∼0.25 and divided into two 1 L flasks, one treated with 1 mM IPTG and the other untreated. Aliquots of 40 ml of cell culture were harvested from both flasks at different time-points.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using an acidic phenol-based method.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
Total RNA were then treated with Turbo-DNAse (Thermo Fisher Scientific) to remove any possible DNA contamination, and then reverse transcribed using SuperScript™ Indirect cDNA Labeling System (Thermo Fisher Scientific) into cDNA. cDNA was aliquoted and labelled with either fluorescent dye 555 or dye 647, and hybridized to microarray slides containing 60 nt oligonucleotides for each coding region according to laboratory protocol.
|
| |
|
| |
| Hybridization protocol |
Equal amount (250 pmol) of labeled cDNA was combined plus hybridization buffer (2X = 50% form amide, 10X SSC, 0.1% SDS). cDNA mix was denatured at 95oC and hybridized 16-18 hours at 42oC to DNA microarray slides which had been prehybridized for at least 30 min at 42oC in 1% bovine serum albumin, 5X SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), washed in water and dried. Following hybridization the slides were washed sequentially in: 2X SSC + 0.1% SDS for 5 min at 42oC, 2X SSC + 0.1% SDS for 5 min at room temperature, 2X SSC for 5 min at room temperature, 0.2X SSC for 5 min at room temperature, and finally dipped in water and spun until dry.
|
| Scan protocol |
Arrays were scanned using a GenePixTM 4000B array scanner (Axon Instruments, Inc.). Raw data files were produced from the scanned images using the GenePix Pro 4.0 software package (GPR files).
|
| Data processing |
Red/green fluorescence intensity values were normalized using the GenePix Pro 4.0 software package such that the ratio of medians of all features was equal to 1
|
| |
|
| Submission date |
Jun 29, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Hualiang Pi |
| E-mail(s) |
[email protected]
|
| Organization name |
Cornell University
|
| Department |
Microbiology
|
| Street address |
125 Wing Dr.
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14850 |
| Country |
USA |
| |
|
| Platform ID |
GPL13726 |
| Series (1) |
| GSE100668 |
The Graded Response of Fur Regulon to Iron Limitation in Bacillus subtilis |
|
