Profiling circRNAs Bound to HuD

circRNAs bound to HuD were isolated from the striatum of HuD-OE mice by RNA immunoprecipitation (RIP) using Dynabeads^® (Thermo Fisher Scientific) coated with mouse monoclonal anti-myc tag antibody (9B11; Cell Signaling Technology Inc.) specifically recognizing myc-tagged HuD transgenic protein expressed in HuD-OE as described before (Bolognani et al., 2010 (link); Zimmerman et al., 2020 (link)). Controls for RIP assays were performed using either non-immune IgG and HuD-OE tissue or the myc-tag antibody and wild type (WT) tissue. Aliquots for both RNA and proteins were taken before (input) and after the IP and circRNAs were quantitated using circRNAs arrays as described below. Immunoprecipitated RNA was extracted with Trizol^® (Invitrogen) according to manufacturer instructions and subjected to either circRNA array or qRT-PCR as described below.

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Dell’Orco M., Oliver R.J, & Perrone-Bizzozero N. (2020). HuD Binds to and Regulates Circular RNAs Derived From Neuronal Development- and Synaptic Plasticity-Associated Genes. Frontiers in Genetics, 11, 790.

Publication 2020

Dynabeads mouse monoclonal anti-myc tag antibody Trizol

Anti antibody Antibody Assays Circrna Hud protein Immunoprecipitation Monoclonal antibody Mouse Proteins Striatum Tissue Transgenic Trizol

Corresponding Organization : University of New Mexico

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Variable analysis

independent variables

Overexpression of myc-tagged HuD transgenic protein in HuD-OE mice

dependent variables

CircRNAs bound to HuD protein
CircRNAs quantitated using circRNA arrays

control variables

Non-immune IgG and HuD-OE tissue
Myc-tag antibody and wild type (WT) tissue

controls

Positive control: non-immune IgG and HuD-OE tissue
Negative control: myc-tag antibody and wild type (WT) tissue

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