whole organism Strain M129 total RNA(including sRNAs)
Growth protocol
Bacterial Strains and culture conditions: Mycoplasma pneumoniae is grown in 75cm2 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 Quiazol lysis reagent from QiagenTM miRNeasy Kit (Cat. Num. 79674). This isolation method is used for RNA extraction and preserves most small of RNAs. For cell lysis, 1.5mL of Quiazol lysis reagent is used per cultivation flask. The purification is done according the manufacturers protocol.
Label
biotin
Label protocol
9μg of total RNA are mixed with 4.5 μg of random hexamers and incubated at 70C for 10min, then transferred on ice. The synthesis included 10μL of SuperScript II Reverse Transcriptase(20 U/μL, Invitrogen, P/N 18064-071), 20 μL of DTT(Invitrogen, P/N 18064-071) 100mM, 40 μL of 5x1st strand buffer(Invitrogen, P/N 18064-071), 5μL of 10mM dNTPs+dUTPs(Affymetrix, P/N 900813) and 2.4 μL of Actinomycin D(0.5mg/mL, Sigma, P/N A1410-5MG) in a total volume of 200 μL at 42C for 1h. Samples then are subjected to RNase treatment of 20min at 37C(30 U Rnase H, Affymetrix, P/N 900813, 60 U Rnase cocktail, Ambion). First-strand cDNA was column purified and fragmented. Fragmentation mixture was composed by 4.8 μL 10x cDNA fragmentation buffer(Affymetrix, P/N 900813), 5.5 μg of cDNA, 1μL APE(1000 U/μL, Affymetrix, P/N 900813), 1 μL UDG (10 U/μ/L, Affymetrix, P/N 900813) in 50 μL at 37C for 60 min. Fragmentation enzymes were inactivated at 93C for 2min.
Hybridization protocol
Labeling, hybridization, staining and scanning is carried out in the EMBL Genomics Core Facility following Affymetrix recommendations
Scan protocol
Labeling, hybridization, staining and scanning is carried out in the EMBL Genomics Core Facility following Affymetrix recommendations
Description
GUELMPM_11_081021.CEL
Data processing
CEL files are parsed together with BPMAP (probe basic annotation) file using AffxParser library implemented in R. Probes complete annotation is carried out using R scripting. NCBI ftp (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/Mycoplasma_pneumoniae)is used as source for probe annotation.
