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SRX25488404: GSM8424732: 35_NRDE3_enri2_early_IP1; Caenorhabditis elegans; ncRNA-Seq
1 ILLUMINA (NextSeq 2000) run: 25.9M spots, 2G bases, 702.3Mb downloads

External Id: GSM8424732_r1
Submitted by: Phillips, Biological Sciences, University of Southern California
Study: Nuclear Argonaute protein NRDE-3 switches small RNA binding partners during embryogenesis coincident with the formation of SIMR granules
show Abstracthide Abstract
RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Further, we observe that many other RNAi factors form foci in embryonic cells distinct from SIMR granules, including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the “SIMR granules”, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during early embryogenesis and switching later in embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions. Overall design: FLAG::NRDE-3 immunoprecipitation from whole C. elegans embryo extract followed by small RNA purification, library preparation, and sequencing
Sample: 35_NRDE3_enri2_early_IP1
SAMN42841797 • SRS22143911 • All experiments • All runs
Library:
Name: GSM8424732
Instrument: NextSeq 2000
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: For immunoprecipitation followed by small RNA sequencing in embryos, ∼500,000 synchronized embryos were sonicated with Fisher Sonifier 550 with a microtip (15s on, 45s off, 10% power, total 2 minutes on time). After sonication, insoluble particulate was removed by centrifugation at 21,000g for 30 minutes. Immunoprecipitation was performed using anti-FLAG Affinity Matrix (Sigma Aldrich, A2220). NRDE-3-bound RNAs were isolated using TRIzol reagent (Thermo Fisher, 15596018), followed by chloroform extraction and isopropanol precipitation. Small RNAs (18 to 30-nt) were size selected on homemade 10% Urea-polyacrylamide gels from total RNA samples. Small RNAs were treated with 5' RNA polyphosphatase (Epicenter RP8092H) and ligated to 3' pre-adenylated adapters with Truncated T4 RNA ligase (NEB M0373L). Small RNAs were then hybridized to the reverse transcription primer, ligated to the 5' adapter with T4 RNA ligase (NEB M0204L), and reverse transcribed with Superscript III (Thermo Fisher 18080-051). Small RNA libraries were amplified using Q5 High-Fidelity DNA polymerase (NEB M0491L) and size selected on a homemade 10% polyacrylamide gel. Library concentration was determined using the Qubit 1X dsDNA HS Assay kit (Thermo Fisher Q33231) and quality was assessed using the Agilent BioAnalyzer. Libraries were sequenced on the Illumina NextSeq2000 (SE 75-bp reads) platform.
Runs: 1 run, 25.9M spots, 2G bases, 702.3Mb
Run# of Spots# of BasesSizePublished
SRR3000908825,904,4042G702.3Mb2024-07-31

ID:
34278631

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