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| Status |
Public on Dec 02, 2019 |
| Title |
Ago25_15 |
| Sample type |
SRA |
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| Source name |
AGO1-IP heads
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Canton-S
circadian timepoints: zt15
temperature: 25C
tissue: fly heads
molecule subtype: small RNA
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| Treatment protocol |
Ago-IP was done as described at Kadener. S et al, Genes Dev 2009, with the following modifications: 200µl of fly heads were homogenized in 700 μL of lysis buffer (30 mM HEPES KOH at pH 7.4, 100 mM KAcetate, 2 mM MgAcetate, 5% glycerol, 0.5% IGEPAL CA-630, 1 mM EGTA, 1 mM DTT, 0.4 U/μL RNase OUT [Invitrogen], 1× EDTA free protease inhibitor [Roche]) using a motorized pellet pestle. The lysates were incubated for 10 min on ice and centrifuged at 21,000 rcf (Max speed) for 15 min at 4°C. 10% supernatant were used as input. For Ago IP, 2µg of anti-AGO-1 antibody (kindly provided by Prof. Mikiko C. Siomi , The University of Tokyo) was incubated for 2 hours at 4°C with rotation. Following incubation with protein G-plus magnetic bead slurry (Dynabeads.ThermoFisher Scientific) for an additional one hour (at 4°C with rotation), the beads were washed five times with 500μL of lysis buffer, with the magnet placed on ice.
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| Growth protocol |
Flies were grown at different temperatures
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was done using TRI Reagent [SIGMA] following manufacturer´s protocol. The precipitated RNAwas taken for library preparation.
Libraries were constructed using NEBNext® Small RNA Library Prep Set for Illumina.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Processing was done using miRExpress pipeline (Wang, W.C., et al., miRExpress: Analyzing high-throughput sequencing data for profiling microRNA expression. BMC Bioinformatics, 2009. 10(1): p. 328.) using miRBase 21 version
Genome_build: miRBase 21
Supplementary_files_format_and_content: txt file with miRNA quantification
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| Submission date |
Dec 19, 2018 |
| Last update date |
Dec 02, 2019 |
| Contact name |
Sebastian Kadener |
| E-mail(s) |
[email protected]
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| Organization name |
Brandeis University
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| Street address |
415 South Street
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| City |
Waltham |
| State/province |
MA |
| ZIP/Postal code |
9190401 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE124134 |
Small RNA seq from Ago1-IP of fly heads at different temperatures and circadian timepoints |
| GSE124136 |
Differences in circadian rhythms in fly at different temperatures |
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| Relations |
| BioSample |
SAMN10616373 |
| SRA |
SRX5165605 |
| 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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