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SRX21162832: GSM7662711: HRDE-1, hrde-2 (adult) - input, replicate 1; Caenorhabditis elegans; ncRNA-Seq
1 ILLUMINA (NextSeq 500) run: 20.6M spots, 1.6G bases, 675.1Mb downloads

External Id: GSM7662711_r1
Submitted by: Phillips, Biological Sciences, University of Southern California
Study: Germ granule association drives small RNA specificity for a nuclear Argonaute protein [sRNA-seq]
show Abstracthide Abstract
RNA interference (RNAi) is a conserved gene silencing process that exists in diverse organisms to protect genome integrity and regulate gene expression. In C. elegans, majority of RNAi components are located in phase-separated perinuclear germ granules, including P granule, Mutator foci, Z granule, and SIMR foci. However, the protein components and function of the newly discovered SIMR foci are unknown. Here we identified HRDE-2 as a SIMR foci component, which interacts with the germline nuclear RNAi Argonaute HRDE-1. We found that unloaded HRDE-1 localizes to SIMR foci and this localization depends on HRDE-2. In addition, HRDE-2 contributes to HRDE-1 small RNAs binding specificity and, in the absence of HRDE-2, HRDE-1 exclusively loads CSR-class 22G-RNAs rather than WAGO-class 22G-RNAs, promotes H3K9me3 deposition on CSR-targets, but does not silence these CSR-target genes. Thus, our study demonstrates that HRDE-2 is critical to ensure correct small RNAs are used to guide nuclear RNA silencing in the C. elegans germline. Overall design: FLAG::HRDE-1 immunoprecipitation from whole worm extract followed by small RNA purification, library preparation, and sequencing
Sample: HRDE-1, hrde-2 (adult) - input, replicate 1
SAMN36714618 • SRS18424816 • All experiments • All runs
Library:
Name: GSM7662711
Instrument: NextSeq 500
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: For immunoprecipitation, ~100,000 synchronized 1-day-old adult animals were collected in IP Buffer (50 mM Tris-Cl pH 7.4, 100 mM KCl, 2.5 mM MgCl2, 0.1% Igapal CA-630, 0.5 mM PMSF, cOmplete Protease Inhibitor Cocktail, and RNaseOUT Ribonuclease Inhibitor), frozen in liquid nitrogen, and homogenized using a mortar and pestle. After further dilution into IP buffer (1:10 packed worms:buffer), insoluble particulate was removed by centrifugation and a sample was taken as “input.” The remaining lysate was used for the immunoprecipitation. Immunoprecipitation was performed at 4°C for 1 hour, then washed at least 3 times in immunoprecipitation buffer. Trizol reagent was added to the remainder of each sample, followed by chloroform extraction, and isopropanol precipitation. Small RNAs (18 to 30-nt) were size selected on denaturing 15% polyacrylamide gels from total RNA samples. Small RNAs were treated with 5' RNA polyphosphatase and ligated to 3' pre-adenylated adapter with Truncated T4 RNA ligase. Small RNAs were then hybridized to the TruSeq reverse transcription primer, ligated to the 5' adapter with T4 RNA ligase), and reverse transcribed with Superscript III. Small RNA libraries were amplified using Q5 High-Fidelity DNA polymerase and size selected on a 10% polyacrylamide gel. Library concentration was determined using the Qubit 1X dsDNA HS Assay kit and quality was assessed using the Agilent BioAnalyzer. small RNA-seq
Runs: 1 run, 20.6M spots, 1.6G bases, 675.1Mb
Run# of Spots# of BasesSizePublished
SRR2542824120,579,4191.6G675.1Mb2023-08-01

ID:
28598166

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