Project description:To obtain translational profiles for all mRNAs, polysome preparations are separated according to their size using a sucrose gradient and the mRNAs in each fraction are identified and quantified with DNA microarrays. Starting with exponentially growing cells, we analyzed 12 polysome fractions using DNA microarrays containing elements for all known and predicted genes of fission yeast. This approach provided data on average numbers of associated ribosomes for most transcripts.
Project description:Genome-wide translational profiling of rng3-65 compared to wild type cells. We used sucrose gradients to separate RNAs according to the number of associated ribosomes (a surrogate for translational efficiency). Preparation of the extracts and fractionation was carried out as described in Lackner et al, 2007 (Mol Cell 26(1):145-55). The fractions were pooled into four groups (1 closest to the top, i.e. not associated with ribosomes and 4 closest to the bottom, i.e., associated with polysomes). RNA was extracted from the pools and the corresponding pools from wild type and mutant cells were directly compared using DNA microarrays. Changes in translation are expected to alter the number of ribosomes associated with specific transcripts, and therefore result in a redistribution of the RNAs across the different fractions.
Project description:The human dataset includes the gene expression profile of CD4+ T cells isolated from blood of healthy controls and plated on TCP in RPMI-1640 containing 10% FCS, Penicillin-Streptomycin (50,000 units-50 mg) and L-glutamine (2 mM). Cells were stimulated for 4 days with 20 ng/ml of IL-1beta, 100 IU/ml of IL-2, 20 ng/ml of IL-6, 20 ng/ml IL-23 plus anti-CD2/3/28 beads at a ratio of 1 bead per 10 cells. RNA samples were isolated using the RNeasy Mini Kit (Qiagen) with on-column DNA digestion. The transcriptional profile was evaluated in three different donors using the HT12v4.1 BeadChip arrays from Illumina. Total RNA obtained from CD4+ T cells exposed to Th17-promoting cytokines.
Project description:Gene Expression Changes in Lymphocytes at Various Times Post-Feline Immunodeficiency Virus Infection:<br> <br> Lymphocytes were cultured for 3 to 5 days with hrIL-2 and Con-A, counted, and prepared at a density of 4.0 x 10e6 cells/ml of media. Approximately 10e7 cells were seeded into eight separate 50 ml centrifuge tubes, and cells were treated with polybrene for 1 hour at 37oC.<br> The cells were washed and infected with 16 TCID50 of USgaB01 for 2 hours, or mock infected with "spent" media. The cells were washed, re-suspended in 10 ml of fresh media, and aliquots were cultured for 1, 2, 4, and 24 hours. Total RNA was isolated at each time<br> point for microarray analysis. Individual aliquots of lymphocytes were infected on 3 separate<br> occasions.
Project description:A defining characteristic of quiescent cells is their low level of gene activity compared to growing cells. Using a yeast model for cellular quiescence, we compared the genome-wide profiles of multiple histone modifications between growing and quiescent cells, and correlated these profiles with the presence of RNA polymerase II and its transcripts. Quiescent cells retained several forms of histone methylation normally associated with transcriptionally active chromatin and had many transcripts in common with growing cells. Quiescent cells also contained high levels of RNA polymerase II, but only low levels of the canonical initiating and elongating forms of the polymerase. The data suggest that the transcript and histone methylation marks in quiescent cells were either inherited from growing cells or established early during the development of quiescence and then retained in this non-growing cell population. This might ensure that quiescent cells can rapidly adapt to a changing environment to resume growth. Immunoprecipitation experiments were carried out for Pol II, H3K36me3, H3K4me3, H3K79me3 and H3 separately using both Log-phase and Quiescent cells. Each ChIP-chip assay was conducted using a control (IN) and in all instances, at least one replicate experiment was performed.