Unknown,Transcriptomics,Genomics,Proteomics

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Lymph node stromal cells: Control siRNA treated vs. Eif4g3 siRNA treated - Total RNA and Heavy polysome fraction

ABSTRACT: Gene expression in the total RNA and heavy polysome fractions of Eif4g3 siRNA treated lymph node stromal cells (LNSCs) compared to control-sIRNA treated samples The objective of this study was to identify genes whose translation are reduced after silencing Eif4g3 (the gene which encodes the translation initiation factor eIF4GII). Genes with reduced translation are expected to have lower expression in RNA samples isolated from heavy polysomes but not in RNA samples isolated from whole cell lysates. LNSCs were grown in 10 cm plates, allowed to reach M-bM-^IM-% 80% confluency and then transfected with 400 pmol of control or Eif4g3 siRNA using Lipofectamine 2000. 7 plates were transfected with control or Eif4g3 siRNA. Total RNA was extracted from cells 48h after transfection. To isolate RNA from the heavy polysome fractions, cells were pooled, lysed and fractionated on a 10% to 60% continuous sucrose gradient. Fractions containing the heavy polysome fractions were pooled. RNA was extracted from these samples and used for microarrays on the Agilent Whole Mouse Genome Microarray Kit, 4M-CM-^W44K 2-color arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Linda Yip

PROVIDER: E-GEOD-39408 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A long-standing view of development is that transcription is silenced in the oocyte until early divisions in the embryo. The point at which major transcription is reactivated varies in different organisms, but is usually after the 2-cell stage. However, this model may not be universal. We used RNA-seq and exploited the protracted development of the parasitic nematode Ascaris suum, to provide a comprehensive time course of mRNA expression, degradation, and translation during early development. Surprisingly, we find that ~4,000 genes are transcribed prior to pronuclear fusion and in the 1-4 cell embryos. Intriguingly, we do not detect maternal contribution of many orthologs of maternal C. elegans mRNAs, but instead find these are newly transcribed in the A. suum zygote prior to pronuclear fusion. Ribosome profiling demonstrates that, in general, early embryonic mRNAs are not stored for subsequent translation, but are directly translated following their synthesis. The role of maternally contributed and zygotically transcribed genes differs between the nematodes A. suum and C. elegans despite the fact that the two nematodes appear to exhibit highly similar morphological patterns during early development. Our study indicates that major transcription can occur immediately after fertilization and prior to pronuclear fusion in metazoa, suggesting that newly transcribed genes appear to drive A. suum early development. Furthermore, the mechanisms used for controlling the timing of the expression of key conserved genes has been altered between the two nematodes, illustrating significant plasticity in the regulatory networks that play important roles in developmental outcomes in nematodes. A total of 28 samples are used for this study. The RNAs for the library construction are derived from 0.5 M-bM-^@M-^S 1 ml of synchronized embryo stages corresponding to a minimum of 0.5 billion embryos derived from > 1,000 female worms. The RNAs for polysome analysis are from pooled fractions of polysome gradient. For each sample, the amount of RNA for all polysome fractions is equivalent to the total RNA from the sample. Thus, the data from polysome profiling and RNA transcriptomes are considered as biological replicates. The level of the RNAs between each neighboring stages during development are considered as the reference for each other. Different sequencing strategies were applied for multiple libraries.

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Lymph node stromal cells: Control siRNA treated vs. Eif4g3 siRNA treated - Total RNA and Heavy polysome fraction

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