Single Cell RNA-Sequencing of RNAi treated Schneider S2 cells
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ABSTRACT:
PROVIDER: PRJEB49369 | ENA |
REPOSITORIES: ENA
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Project description:Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)Âdependent, 3'-end modified siRNAs. To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidÂbased functions and include Argonaute-2 (AGO2) itself. Experiment Overall Design: Drosophila Schneider cells (S2) were treated with dsRNA against GFP for 8 days. The treatment was done in duplicate. Total RNA was extracted from the dsRNA treated cells using trizol. Experiment Overall Design: Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3¹ Amplification One-Cycle Target labeling kit according to manufacturer¹s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
2008-06-20 | E-GEOD-11370 | biostudies-arrayexpress
Project description:In order to identify interaction partner of the Drosophila melanogaster TFIIA protein, we have immunoprecipitated an endogenously 3xFLAG-AID tagged TFIIA-L from Drosophila Schneider S2 cells
2023-02-16 | PXD028094 | Pride
Project description:Proteomic analysis of S2 cells subjected to RNAi with either HOW(S) specific RNAi duplex or non-specific control
2026-02-09 | PXD070902 | Pride
Project description:2 million cells were seeded in a 2 ml medium containing 58 µg of total dsRNA. For coRNAi treatments 28 µg of each single dsRNA were used. After treatment cells were left to incubate 4 d at 25 °C, and plates were sealed with Parafilm to avoid medium evaporation. 8 samples were prepared with the following dsRNA treatment combinations: RLUC/RLUC x 2, CSN5/RLUC, CSN8/RLUC, CDK2/CSN5, CDK2/CSN8, CDK2/RLUC 2x . dsRNAs were taken from the HD3-dsRNA-library. Two dsRNAs per gene were pooled. The single-cell transcriptomic sequencing experiment was performed with 10x Genomics technology according to the manufacturer’s protocol.
2022-01-03 | E-MTAB-11196 | biostudies-arrayexpress
Project description:Background: The tissue-specific response to steroid hormones remains unclear even in model organisms. The mechanism is proposed to be based on the selective binding of nuclear hormone receptors to regulatory sites that are primed by tissue-specific factors. But studies directly describing the cooperative work of nuclear receptors with tissue-specific transcription factors remain sparse. Earlier, GATA family factor SRP/dGATAb was proposed to prime regulatory sites in S2 Schneider cells for their activation by 20-hydroxyecdysone (20E), the main steroid hormone of the Drosophila development. Highly specific expression of SRP/dGATAb in Drosophila tissues makes it a suitable candidate for a factor enabling tissue-specific response to 20E. Results: The role of SRP/dGATAb in the response of S2 Schneider cells to 20E was determined using SRP/dGATAb depletion via RNA interference. Combination RNA-Seq with ChIP-Seq analysis helped identify the primary targets of SRP/dGATAb whose transcription is induced in response to 20E treatment and whose loci contain EcR and SRP/dGATAb binding sites. SRP/dGATAb depletion was found to alter transcription of different 20E-induced genes in opposite ways. For 20E-activated genes whose transcription was decreased upon depletion, SRP/dGATAb was found to control active regulatory sites marked by the H3K27Ac and enriched with SRP/dGATAb motifs. In this group SRP/dGATAb depletion reduced EcR binding level at sites co-bound with EcR and SRP/dGATAb, demonstrating a priming role for GATA family protein. In contrast, for 20E-activated SRP/dGATAb-suppressed genes whose transcription was increased upon SRP/dGATAb depletion, SRP/dGATAb and EcR co-bound sites showed unchanged H3K27Ac levels and EcR binding. The overall level of H3K27 acetylation at the loci of the second group of genes showed increase upon SRP/dGATAb depletion. Conclusions: Obtained data showed a positive role of SRP/dGATAb in 20E-inducible transcription in S2 Schneider cells for some genes. SRP/dGATAb worked as a priming transcriptional factor for 20E-inducible regulatory sites controlling EcR recruitment and chromatin acetylation. But for another group of 20E-inducible genes, SRP/dGATAb had negative impact on transcription. Its recruitment through the protein-protein interactions restrained activity of the regulatory sites.
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