Project description:Total RNA was isolated from wild-type adult flies (yellow white) and from embryos (cs, 0-16h) with TRIZOL reagent. 12 microgram of each total RNA were labeled with fluorescent dyes, following cDNA synthesis with amino-allyl dUTP in addition to the four natural dNTPs using a 1:1 mixture of oligo(dT) and random nonamer primers. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: all_pairs
Project description:To dissect how diurnal rhythms affect key functions such as transcription or chromatin remodeling, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC-based MS. Protein extracts from isotope labelled mice liver nuclei were used as a reference and mixed with extracts from animals collected every 3h for 45h total. Total protein levels were analysed together with phosphopeptides after enrichment (see separate dataset for phospho data).
Project description:Methodologies for untargeted metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) were developed and applied to lipid-depleted methanolic extracts of soybeans seeds obtained from four near isogenic soybean lines (NILs) that differed in phytate content. The four lines differed in mutations for genes encoding two highly homologous multi-drug resistant proteins (MRPs). The double mutant exhibited the low phytate/emergence phenotype whereas the other three NILs, namely the two single mutants and the wild type, did not. Lipid-depleted aqueous methanol extracts were analyzed by orthogonal chromatographic separations (reversed-phase and hydrophilic interaction) in both positive and negative ionization modes. Principal component analysis (PCA) of the LC-MS data clearly separated the low phytate line from the other three, with the major metabolite differences residing in the soyasaponin composition. Group A soyasaponins containing C22-terminated acetylated glucosides were of much lower concentration in the low phytate line, which favored terminally acetylated xyloside-based soyasaponins (soyasaponin A4, A5 and A6). Differing levels of the allergen Gly m 1 were also detected.
Project description:Four-day old seedlings of Arabidopsis wildtype Col-0, T-DNA insertional mutant ataf2-2, and overexpression line ATAF2ox-3 were collected for total RNA extraction. Three biological replicates were prepared for each genotype.
Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs
Project description:To compare four- and seven-day-old Arabidopsis thaliana plasmodesmata proteome, the identification and the relative amount of proteins in plasmodesmata, cell wall and total cell extracts were performed using fractions purified from four- and seven-day-old liquid culture cells of A. thaliana. The relative amount for each protein was determined with a label-free quantification method. Four biological replicates of each fraction were used for quantification.
Project description:RNA co-immunoprecipitations with C-terminal protein A-tagged She proteins. One liter of cells were cultured at 30 degrees in YPAD medium and collected during exponential growth by centrifugation. Cells were washed twice and broken mechanically with glass beads. Extracts were incubated with IgG-agarose beads (Sigma). The beads were washed four times, and She proteins were released from the beads by cleavage with TEV-protease (Invitrogen). RNA was isolated by phenol/chloroform extraction and isopropanol precipitation from TEV eluates, which corresponds to the purified fraction, and from extracts (input). Both RNA samples, input and purified, were reverse transcribed and labeled with the fluorescent dyes Cy3 and Cy5 (Amersham), respectively. The samples were mixed and competitively hybridized to yeast DNA microarrays containing all yeast genes Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set