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RIP-seq of livers in C57BL/6J mice with antibodies against Cnot3, Ago2, and BRF1

ABSTRACT: RIP-seq of livers in C57BL/6J mice with antibodies against Cnot3, Ago2, and BRF1

PROVIDER: PRJEB27420 | ENA |

REPOSITORIES: ENA

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RIP-seq of livers in C57BL/6J mice with antibodies against Cnot3, Ago2, and BRF1

Project description:Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. The CCR4-NOT complex is the major deadenylase in mammals. Interaction of Cnot1 with RNA binding proteins, including the miRNA-induced silencing complex (miRISC), which includes Argonaute (Ago) and GW182 as core proteins, and the TTP family of AU-rich element (ARE)-binding proteins (TTP and BRF1/2), recruits the CCR4-NOT complex to the 3’-untranslated regions (3’-UTR) of mRNAs, leading to their degradation. This experiment aimed to determine the binding affinity of the CCR4-NOT complex, Ago2, and BRF1 with their target mRNAs in liver. Liver isolated from C57BL/6J wild-type mice at 8 weeks of age was solubilized in TNE buffer for 30 min at 4ºC. Lysate was incubated with antibodies against Cnot3, Ago2, and BRF1 for 1 hr at 4ºC, and then incubated with Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II. 100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep. 150 base-pair pair-end read RNA-seq was performed with Hiseq 3000/4000 PE Cluster Kit and Hiseq 3000/4000 SBS Kit on Hiseq4000.

2020-03-30 | E-MTAB-6941 | biostudies-arrayexpress

Transcriptome wide analysis of Cnot3 target mRNA in mouse hearts

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Differential Argonaute-2 RNA immunoprecipitation of control cells versus cells overexpressing miR-146a followed by deep sequencing (Ago2 RIP-seq)

Project description:We undertook an unbiased approach based on differential Argonaute-2 (Ago2) RNA immunoprecipitation followed by deep sequencing (Ago2 RIP-seq) to unravel the molecular mechanism(s) underlying miR-146a function, namely the relevant mRNA target(s). Ago2 is a crucial protein that engages with both miRNA and its mRNA target(s) in the RISC complex. To overcome the problem of lack of commercially available high affinity antibodies against Ago2, we took advantage of a genetically modified mouse in which endogenous Ago2 is replaced with a N-terminal tagged version of the protein (3xFLAG- 6xHIS), thus allowing stringent purification (i.e., with low unspecific background) of the RNA molecules bound to Ago2 (upon UV-crosslinking), followed by RNA-sequencing. In order to enrich for miR-146a targets, we transduced purified CD3+ T cells (we could not use γδ T cells due to insufficient cell numbers for this approach) with a miR-146a-expressing retrovirus (with~12-fold overexpression of miR-146a). We thereby identified 225 putative mRNAs after differential Ago2-RIP-seq, from which 96 (42,7%) had a predicted miR-146a binding site in their 3’ UTR region.

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Identification of KSHV, EBV and cellular miRNA targets in human B-cells using RIP-Chip

Project description:RIP-Chip was performed on DG75-eGFP, DG75-10/12, BCBL-1, BL41, BL41 B95.8 and Jijoye using anti-human Ago2 (11A9) antibodies. Anti-BrdU antibodies were used as controls for DG75-eGFP, DG75-10/12 and BCBL-1. Total RNA was used as control for BL41, BL41 B95.8 and Jijoye. Samples were analyzed on Affymetrix Gene ST 1.0 Arrays (2 independent biological replicates / sample) KSHV, EBV and cellular miRNA targets were determined by RIP-Chip using monoclonal antibodies to human Ago2

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Gene expression profile of Cnot3(+/+) & Cnot3 (∆/∆) pro-B cells

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Project description:Argonaute (Ago) proteins associate with microRNAs (miRNAs), which guide them to complementary target mRNAs resulting in gene silencing. RNA coimmunoprecipitations (RIP) were performed with Ago2 and Ago2 mutants. The precipitated mRNAs were analyzed using one Affymetrix microarrays to compare the mRNA preciptation rates of mutants vs Ago2 wild type.

2017-06-22 | GSE92698 | GEO

Ago2[414] Ago1 Immunoprecipitation

Project description:modENCODE_submission_2746 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Keywords: RIP-seq EXPERIMENT TYPE: RIP-seq. BIOLOGICAL SOURCE: Strain: Ago2 414; Developmental Stage: Adult Female; Genotype: w;;ago2[414]; Sex: Female; EXPERIMENTAL FACTORS: Adult_ovaries

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