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Series GSE52799 Query DataSets for GSE52799
Status Public on Jun 12, 2014
Title Polysome profiling and ribosome footprinting of Drosophila mature oocyte and activated egg
Organism Drosophila melanogaster
Experiment type Expression profiling by high throughput sequencing
Summary 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.
 
Overall design Lysates of hand-dissected Drosophila mature oocytes (containing ~540 μg 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 manufacturer’s instructions. mRNA-seq samples were prepared from 1 μg 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 μg 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 ≥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).
 
Contributor(s) Kronja I, Eichhorn S, Yuan B, Bartel DP, Orr-Weaver TL
Citation(s) 24882012
Submission date Nov 27, 2013
Last update date May 15, 2019
Contact name Iva Kronja
Organization name Whitehead Institute for Biomedical Research
Lab Terry Orr-Weaver
Street address 9 Cambridge Center
City Cambridge
State/province MA
ZIP/Postal code 02142
Country USA
 
Platforms (1)
GPL13304 Illumina HiSeq 2000 (Drosophila melanogaster)
Samples (54)
GSM1276347 input_(unfractionated lysate)_mature_oocyte_replicate_1
GSM1276348 RNPs_fraction_mature_oocyte_replicate_1
GSM1276349 40S_fraction_mature_oocyte_replicate_1
Relations
BioProject PRJNA230057
SRA SRP033366

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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE52799.txt.gz 264 b (ftp)(http) TXT
GSE52799_Overlap_samples_43_54.txt.gz 419.4 Kb (ftp)(http) TXT
GSE52799_RAW.tar 3.3 Mb (http)(custom) TAR (of TXT)
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record
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