|
| Status |
Public on Nov 20, 2006 |
| Title |
C. novyi-NT spore Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
C. novyi-NT spore
|
| Organism |
Clostridium novyi NT |
| Characteristics |
C. novyi-NT spore
|
| Growth protocol |
The bacteria were cultured in sporulation medium for at least two weeks to ensure maximum yield of mature spores. Mature spores were purified through two consecutive continuous Percoll gradients followed by four washes and resuspensions in phosphate-buffered saline (PBS). Purity of the spore preparations was determined to be >99.9% using phase contrast microscopy as well as by staining with malachite green and eosin Y (http://pb.merck.de/servlet/PB/show/ 1235100/115942en.pdf).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Vegetative bacteria or spores were collected by centrifugation and homogenized by vortexing in RNAwiz buffer (RiboPureTM-Bacteria kit, Ambion, Austin, TX) containing 250 µL of Zirconia beads (Biospec, Bartlesville, OK) for 10 minutes at 4°C, followed by a 5-min break. This cycle was repeated 12 times to ensure disruption of the spores (as assessed by phase contrast microscopy and RNA yield). RNA was purified with the RiboPureTM-Bacteria kit (Ambion) following the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
total RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen). The first-strand cDNA was fragmented by DNase I (Promega, Madison WI) and labeled with biotinylated ddATP through a terminal transferase end-labeling reaction.
|
| |
|
| Hybridization protocol |
Hybridization with the end-labeled samples was carried out at 45°C for 16 hours in 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20 and 10.5% glycerol.
|
| Scan protocol |
After washing, the arrays were stained with Cy3-Streptavidin, followed by scanning with an Axon 4000B scanner.
|
| Description |
The features were extracted by using the NimbleScan software.
|
| Data processing |
Data was analyzed with the Bioconductor project using quantile normalization (see:Bolstad B, Irizarry R, Astrand M, Speed T (2003) A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics 19 (2), p185-193) and gene calls generated using the RMA algorithm (Robust multichip average described in Biostatistics 2003 Apr;4(2):249-64 ).
|
| |
|
| Submission date |
Oct 18, 2006 |
| Last update date |
Nov 20, 2006 |
| Contact name |
Xin Huang |
| E-mail(s) |
xhuang8@jhmi.edu
|
| Organization name |
Johns Hopkins University
|
| Department |
Oncology
|
| Street address |
1650 Orleans Street, CRB I, Room 520
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL4463 |
| Series (1) |
| GSE6087 |
Transcriptomes of Clostridium novyi-NT |
|
