|
| Status |
Public on Aug 12, 2016 |
| Title |
Xylose treated WT labeled with 647 vs Depletion Strain labeled with 555 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Treated Depletion strain
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: Depletion strain
|
| Treatment protocol |
The wild type and depletion strains were grown separately in liquid LB medium to OD600 ~0.4, and then each diluted 100-fold into two 250 ml flasks each containing fresh 50 ml LB medium. After 1.5 hours of initial growth, one of the two flasks of each strain was supplemented with 50% xylose to a final concentration of 2%, while the other flask was left untreated as control. After another 2.5 hours of growth, cells were collected and total RNA was extracted using phenol-chloroform based method.
|
| Growth protocol |
The wild type and depletion strains were grown separately in liquid LB medium to OD600 ~0.4, and then each diluted 100-fold into two 250 ml flasks each containing fresh 50 ml LB medium. After 1.5 hours of initial growth, one of the two flasks of each strain was supplemented with 50% xylose to a final concentration of 2%, while the other flask was left untreated as control. After another 2.5 hours of growth, cells were collected and total RNA was extracted using phenol-chloroform based method.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using phenol-chloroform based method. Total RNA was quantified and quality of RNA was tested using Bio-analyzer. RNA quality numbers for all four RNA samples are 10 out of 10.
|
| 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 |
Treated WT
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: wildtype
|
| Treatment protocol |
The wild type and depletion strains were grown separately in liquid LB medium to OD600 ~0.4, and then each diluted 100-fold into two 250 ml flasks each containing fresh 50 ml LB medium. After 1.5 hours of initial growth, one of the two flasks of each strain was supplemented with 50% xylose to a final concentration of 2%, while the other flask was left untreated as control. After another 2.5 hours of growth, cells were collected and total RNA was extracted using phenol-chloroform based method.
|
| Growth protocol |
The wild type and depletion strains were grown separately in liquid LB medium to OD600 ~0.4, and then each diluted 100-fold into two 250 ml flasks each containing fresh 50 ml LB medium. After 1.5 hours of initial growth, one of the two flasks of each strain was supplemented with 50% xylose to a final concentration of 2%, while the other flask was left untreated as control. After another 2.5 hours of growth, cells were collected and total RNA was extracted using phenol-chloroform based method.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using phenol-chloroform based method. Total RNA was quantified and quality of RNA was tested using Bio-analyzer. RNA quality numbers for all four RNA samples are 10 out of 10.
|
| 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 amounts (100-150 pmol) of labeled cDNA were combined plus hybridization buffer (2X = 50% formamide, 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 |
Aug 11, 2016 |
| Last update date |
Aug 12, 2016 |
| Contact name |
John Daniel Helmann |
| E-mail(s) |
[email protected]
|
| Phone |
607 255 6570
|
| Organization name |
Cornell University
|
| Department |
Microbiology
|
| Street address |
372 Wing Hall, Cornell University
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL7420 |
| Series (1) |
| GSE85492 |
Depletion of undecaprenyl pyrophosphate phosphatases (UPP-Pases) disrupts cell envelope biogenesis in Bacillus subtilis |
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