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| Status |
Public on Mar 27, 2020 |
| Title |
mid_cyt_RNA_B |
| Sample type |
SRA |
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| Source name |
Embryo
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental window: 8-16 hours
developmental stage: mid
replicate: replicate B
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| Treatment protocol |
After removing pre-laying plates, embryos were flash frozen, turned into powder using a pre-chilled pestle and mortar and homogenized in Lysis Buffer at 4°C for 20 min.
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| Growth protocol |
Wild-type Drosophila melanogaster flies (Oregon-R) were grown in standard cages with molasses egg-laying plates containing yeast paste.
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| Extracted molecule |
total RNA |
| Extraction protocol |
We purified mRNAs in polysomes, away from monosomes (80S), ribosomal subunits (40S, 60S). Footprinting was performed overnight at 4°C with RNaseI (Invitrogen), stopped with SUPERase·In TM RNase inhibitor (Invitrogen) and precipitated. mRNA from total cytoplasmic lysate, was purified using oligo (dT) Dynabeads (Invitrogen) and fragmented by alkaline hydrolysis.
28-34 nt ribosome footprints and 50-80 nt mRNA fragments were gel purified and prepared as previously described (Ingolia et al, 2012; Ingolia et al, 2009; Ingolia et al, 2011) for Next Generation Sequencing.
Libraries were sequenced on Illumina NextSeq and MiSeq machines with 50, 75 and 150bp SingleEnd read protocol.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
cytoplasmic poly-A RNA
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| Data processing |
Library strategy: Ribo-Seq
Sequencing reads were clipped, trimmed and aligned to an rRNA, snoRNA, snRNA & tRNA reference using Bowtie, discarding the alignments and collecting unaligned reads (Ingolia et al 2012).
Unaligned reads were mapped to the whole genome of Drosophila melanogaster FlyBase (Release 6.13) using HISAT2, default setiings.
Profiles of ribosome footprints were constructed by quantifying the number of footprint reads aligned at each position within the feature (ORF) of interest. Ribosome density was computed by scaling read counts for each feature-by-feature length and by the total number of genome-aligned reads, using Rsamtool package (http://www.bioconductor.org/packages/release/bioc/html/Rsamtools.html).
For framing analyses, all ribosome footprints were aligned to a custom transcriptome assembly consisting of every ORF studied in its -18 and +15 mRNA context.
For each ORF, the number of reads per frame was counted using the R package "riboSeqR", and the probability of randomly obtaining framing as good or better that the observed was calculated using the R expression "pbinom([framed_reads]-1, [all_reads], 1/3, lower.tail=FALSE)"
Genome_build: FB2016_05 Dmel (Release r6.13)
Supplementary_files_format_and_content: Summary of ribosome density (expressed as RPKM) , framing (expressed as a p-value derived from a binomial test), transcription, ribosome-binding and translation calls, as well as Z-score analysis of translational regulation for annotated canonical protein-coding genes, short coding-sequences (shortCDSs) and upstream ORFs.
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| Submission date |
Mar 27, 2020 |
| Last update date |
Mar 29, 2020 |
| Contact name |
Juan Pablo Couso |
| Organization name |
Centro Andaluz de Biologia del Desarrollo
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| Street address |
Carretera de Utrera, Km 1
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| City |
Seville |
| ZIP/Postal code |
41013 |
| Country |
Spain |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE147619 |
Developmental regulation of Canonical and small ORF translation from mRNAs |
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| Relations |
| BioSample |
SAMN14467019 |
| SRA |
SRX8010218 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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