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A massively parallel reporter assay for RNA localization in neurons

ABSTRACT: A massively parallel reporter assay for RNA localization in neurons

PROVIDER: PRJNA723817 | ENA |

REPOSITORIES: ENA

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A massively parallel reporter assay for RNA localization in neurons

Project description:We performed a massively parallel reporter assay for RNA localization in two mouse neuronal cell lines, CAD and Neuro-2a. We transfected a library of around 50,000 different 3'UTR reporters (cloned downstream of GFP) into cells grown on transwell plates and collected soma and neurite fractions separately. RNA was extracted and cDNA synthesized, followed by targeted amplification of the reporter mRNA and Illumina sequencing.

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Project description:We performed a Massively Parallel Reporter Assay (MPRA) to screen >30,000 human-specific substitutions in ChIP-seq-identified Human Gain Enhancers (HGEs) and Human Accelerated Regions (HARs), highly conserved non-coding regions that show accelerated sequence evolution in humans. After comparing human and chimpanzee reference alleles, we used a second MPRA to deconvolute individual substitutions within differentially active enhancers from substitutions in the same fragment and from other variants (human segregating variants or chimpanzee-specific variants) to isolate their specific effects on enhancer activity.

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Systematic dissection of regulatory motifs in 2,000 predicted human enhancers using a massively parallel reporter assay

Project description:We employ a massively parallel reporter assay (MPRA) to measure the ex vivo activities of hundreds of K562 and HepG2 enhancers with known transcription factor motif instances. For seven selected motifs that correspond to known or predicted activators and repressors in the two cell types, we make directed modifications of the bases corresponding to these motifs and observe the changes in enhancer activity. Reporter mRNA-seq from HepG2 and K562 cells transfected with a ~55,000-plex MPRA plasmid pool containing 5,418 mutated human enhancer sequences, each linked to 10 distinct 10-nt tags. The reporter mRNA tags facilitate quantitation of their abundances. The same tags were also sequenced from the transfected MPRA plasmid pool to facilitate normalization to plasmid copy numbers.

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