|
| Status |
Public on Jun 17, 2011 |
| Title |
112 liaR with RAM rep. 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
liaR/+RAM
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: CU1065 liaR deletion
genotype/variation: liaR deletion
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were grown to mid-log phase (OD600 0.4) and split into 2 flasks. One flask was treated with 5 µg/ml ramoplanin for 10 minutes. Total RNA was isolated using the RNeasy Mini Kit (Qiagen Sciences, Maryland) and treated with TURBO DNA-freeTM (Ambion). RNA concentrations were quantified and cDNA was synthesized from 10 µg total RNA and differentially labeled according to manufacturer’s instructions with Cy3 (combined reference RNA) and Cy5 (sample) (Amersham). Before and after labeling for 1 h at room temperature) cDNA was purified using the Qiagen MinElute PCR Purification Kit (Qiagen, Maryland).
|
| Label |
Cy5
|
| Label protocol |
cDNA synthesis was performed using the SuperScriptTM Plus (Invitrogen) as per the manufacturer’s instructions using 10 microgram of total RNA. Total cDNA was labeled for 1h Cy3/5 Dyes to amino-allyl cDNA.
|
| |
|
| Channel 2 |
| Source name |
WT+/-RAM, liaR +/-RAM (pooled reference RNA)
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: CU1065
genotype/variation: wild-type, liaR deletion (pooled)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were grown to mid-log phase (OD600 0.4) and split into 2 flasks. One flask was treated with 5 µg/ml ramoplanin for 10 minutes. Total RNA was isolated using the RNeasy Mini Kit (Qiagen Sciences, Maryland) and treated with TURBO DNA-freeTM (Ambion). RNA concentrations were quantified and cDNA was synthesized from 10 µg total RNA and differentially labeled according to manufacturer’s instructions with Cy3 (combined reference RNA) and Cy5 (sample) (Amersham). Before and after labeling for 1 h at room temperature) cDNA was purified using the Qiagen MinElute PCR Purification Kit (Qiagen, Maryland).
|
| Label |
Cy3
|
| Label protocol |
cDNA synthesis was performed using the SuperScriptTM Plus (Invitrogen) as per the manufacturer’s instructions using 10 microgram of total RNA. Total cDNA was labeled for 1h Cy3/5 Dyes to amino-allyl cDNA.
|
| |
|
| |
| Hybridization protocol |
Labeled cDNA samples were combined plus hybridization buffer (2X = 50% formamide, 10X SSC, 0.1% SDS). cDNA mix was denatured at 100oC and hybridized overnight at 42oC to DNA microarray slides (oligos from Sigma Genosys #BACLIB96; ultraGAPS coated slides Corning #40016) which had been prehybridized for at least 45 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: 1X SSC + 0.2% SDS for 4 min at 42oC, 0.1X SSC + 0.2% SDS for 4 min at room temperature, 0.1X SSC for 4 min at room temperature, 0.1X SSC for 4 min at room temperature, 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 3.0 software package (GPR files).
|
| Data processing |
Red/green fluorescence intensity values were normalized using the GenePix Pro 3.0 software package such that the ratio of medians of all features was equal to 1. Fold changes equal to average of ramoplanin treated/untreated samples (635) / average of combined reference RNA (532). Ave represents the average of medians for duplicate spots (minus median of background).
|
| |
|
| Submission date |
Jun 15, 2011 |
| Last update date |
Jun 17, 2011 |
| Contact name |
John D. Helmann |
| E-mail(s) |
[email protected]
|
| Phone |
607 255 6570
|
| Organization name |
Cornell University
|
| Department |
Microbiology
|
| Street address |
372 Wing Hall
|
| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL13726 |
| Series (2) |
| GSE30002 |
Bacillus subtilis CU1065 ramoplanin stimulon |
| GSE30003 |
The GntR family repressor YtrA responds to cell wall antibiotics |
|
