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Status |
Public on Aug 19, 2015 |
Title |
Individual-nucleotide resolution CLIP and HTS with GFP-Aub in D. melanogaster 0-2h oskar54 embryos |
Sample type |
SRA |
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Source name |
embryos
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Organism |
Drosophila melanogaster |
Characteristics |
Stage: 0-2h embryos genotype: osk54 UASp-GFP-Aub/ osk54 nos-Gal4 antibody: rabbit polyclonal anti-GFP (A-6455, Invitrogen)
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Treatment protocol |
embryos were UV-irradiated, three times five minutes at 254 nm in a BIO-LINK
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Extracted molecule |
total RNA |
Extraction protocol |
RNA-protein complexes were transferred to nitrocellulose. After cutting out the region of nitrocellulose containing RNA-Aub complexes, RNAs were removed from the membrane by Proteinase K digestion. The RNAs were reverse transcribed using oligonucleotides that contained two adapter regions separated by a restriction site and a barcode region containing a 3 nt experiment-specific barcode and a 4 nt random barcode to mark individual cDNA molecules. cDNA size selection, circularization and linearization was performed as described earlier (Konig et al., 2010, 2011). Linearized cDNAs were then PCR-amplified using primers complementary to the adaptor regions.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2000 |
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Description |
iCLIP-Seq as described by König et al. (König et al; Nat Struct Mol Biol 17:909-915, 2010)
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Data processing |
.fastq files were adapter 'CACTCGGGCACCAAGGACAGGAATG' clipped using cutadapt. After adapter removal, the first 4 bases (corresponding to random barcode) were trimmed and remaining sequences were mapped on D.melanogaster genome allowing up to 2 mismatches for reads mapping to a single genome location and perfect match for reads mapping to more than one location. Reads with identical random barcode, strand, identical 5’ and 3’ end coordinates were collapsed. For small RNA-seq, sequenced reads were stripped of the adapter (CTGTAGGCACCATCAA) in the 3' end and the retrieved small RNA reads were mapped to the genome sequence of Drosophila melanogaster (release 5) using Bowtie, with up to one mismatch (-v 1 -a --best --strata). Libraries were then annotated according to reference databases containing ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), microRNAs (miRNAs), transcripts, small nuclear RNAs (snRNAs) and transposable element sequences. For piRNA identification, we selected reads that were 23-29nt in length and not annotated either as abundant cellular species (tRNAs, rRNAs, snoRNA and snRNA) nor as miRNAs. For iCLIP-seq, the sample- and random barcode sequence were removed from 5’ end following linker sequence trimming at the 3’ end. Trimmed sequence reads were aligned to the genome sequence of Drosophila melanogaster using Bowtie, with up to 2 mismatches for unique mapper reads and 0 mismatch for multi mapper reads. Genome_build: dm5 Supplementary_files_format_and_content: For small RNA-seq, tab-delimited texts containing mapped non-redundant sequences (first column) and corresponding count in the library (second column)
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Submission date |
Jul 10, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Simonelig Martine |
E-mail(s) |
martine.simonelig@igh.cnrs.fr
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Organization name |
Institut de Genetique Humaine - CNRS
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Department |
Genetics and Development
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Lab |
mRNA Regulation and Development
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Street address |
141, rue de la Cardonille
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City |
MONTPELLIER |
ZIP/Postal code |
34396 |
Country |
France |
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Platform ID |
GPL13304 |
Series (2) |
GSE70777 |
Aubergine iCLIP reveals direct regulation of mRNAs involved in germ cell development by piRNAs in the early embryo |
GSE70778 |
Aubergine iCLIP and expression |
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Relations |
BioSample |
SAMN03854471 |
SRA |
SRX1091848 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1818087_iCLIP_GFP-Aub_osk-_embryo0_2h_total_Dm.bedgraph.gz |
15.5 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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