|
| Status |
Public on Dec 11, 2009 |
| Title |
Gentamycin_4 |
| Sample type |
RNA |
| |
|
| Source name |
80 ug/mL of Gentamycin were added 1 hour before lysis.
|
| Organism |
Mycoplasmoides pneumoniae M129 |
| Characteristics |
Strain M129
whole organism
|
| Growth protocol |
Bacterial Strains and culture conditions: Mycoplasma pneumoniae is grown in 150 cm2 tissue culture flasks with 50mL of modified Hayflick medium with the following composition. The basic medium consisted of 18.4g of PPLO broth, 29.8g of HEPES,10 g glucose, 5mL of 0.5% phenol red, and 35mL of 2N NaOH per liter. Horse serum and penicillin were included to a final concentration of 20% and 100 U/mL, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After growth , surface-attached cells are washed once with phosphate-buffered saline (PBS; 0.15M NaCl, 10mM sodium phosphate, pH 7.4) and immediately lysed in the cultivation flask by adding RLT buffer from the QiagenTM RNeasy Plus Mini Kit (Cat. Num. 74134). This isolation method is used for RNA extraction as it removes most RNAs smaller than 200 bases, thus preventing the synthesis of cDNA from tRNA. For cell lysis, 2mL of RLT buffer in presence of 20μL β-mercaptoethanol was used per cultivation flask. The purification is done according the manufacturers protocol.
|
| Label |
Cy5
|
| Label protocol |
9μg of total RNA were used so as to carry out the reverse transcription using SuperScriptTM Indirect cDNA Labeling System from Invitrogen. This kit was used according manufacturer indication but two modifications were introduced in the protocol. Reverse transcription was carried out at 37ºC instead of 46ºC and the set of random hexamers (2uL of 2.5ug/uL) was used instead of polyT 20mers.
|
| |
|
| Hybridization protocol |
Hybridization and Scanning (Axon GenePix 4000) has been carried out in the Genomic Core Facility of EMBL-Heidelberg.
|
| Scan protocol |
Hybridization and Scanning (Axon GenePix 4000) has been carried out in the Genomic Core Facility of EMBL-Heidelberg.
|
| Description |
2007-03-22_MP-61_0635.gpr
|
| Data processing |
After background subtraction quantile (Boltad 2003) normalization was done using the bioconductor package marray (Yang 2005)
|
| |
|
| Submission date |
Dec 17, 2008 |
| Last update date |
Dec 17, 2008 |
| Contact name |
Marc Güell |
| E-mail(s) |
marc.guell@crg.es
|
| Organization name |
Centre for Genomic Regulation
|
| Department |
Systems Biology
|
| Lab |
Design of Biological Systems
|
| Street address |
C/ Dr.Aiguader 88
|
| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7822 |
| Series (2) |
| GSE14015 |
Mycoplasma pneumoniae expression profiling |
| GSE14019 |
Transcriptome/Expression analysis in Mycoplasma pneumoniae |
|
