7 protocols
ID_REF = VALUE = LOESS normalized log2 ratios (Cy3/Cy5) corresponding to 7/0 min or 45/0 min or 90/0 min.
LOESS normalization with Limma package for Bioconductor in R was applied for dye-bias correction.
Transcriptome: Total RNA was isolated by the hot phenol method (Janzon et al., 1986). After DNaseI treatment, RNA was purified by phenol-chloroform-isoamylalcohol and chloroform-isoamylacohol. RNA was further purified by RNeasy®MinElute™ spin columns (Qiagen). Translatome: Polysome fractions were collected by sucrose density gradient centrifugation and polysomal RNA isolated by the hot phenol method. RNA was further purified by RNeasy®MinElute™ spin columns (Qiagen).
Scanned on an Agilent High Resolution Microarray Scanner. Images were quantified using Agilent Feature Extraction Software.
The ULS Fluorescent Labeling Kit for Agilent arrays (Kreatech) was used for RNA labeling and fragmentation.
R. sphaeroides WT 2.4.1 was grown under aerobic conditions (100% oxygen) to an OD660 of 0.4 and stressed with 0.2 µM methylene blue in the presence of 800 W m-2 high light
RNA of three independent experiments of Rhodobacter sphaeroides WT 2.4.1 at 7/45/90 min and 0 min were pooled and hybridized to one array. Gene chip hybridization and scanning were performed according to the specifications from Agilent.