Metabolomics,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:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).

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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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