cox8 egfp mcherry coding region  (Addgene inc)

Journal: Nature

Article Title: Programmable RNA sensing for cell monitoring and manipulation

doi: 10.1038/s41586-022-05280-1

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: 39 , Extended Data Fig 1 g , ​ ,h h , pCAG-spacer-sesRNA-2a-eGFP , Spacer sequence was from CasRx coding regions of Addgene# 109049, different length was used from ATG start as indicated.

Techniques: Construct, Sequencing, Amplification

Journal: Nucleic Acids Research

Article Title: A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

doi: 10.1093/nar/gkac806

Figure Lengend Snippet: A massively parallel reporter assay for RNA localization in neurons. ( A ) Experimental outline. An oligonucleotide library (containing common primers for amplification (2 × 18 nt), a 5′ barcode (12 nt) and a 150 nt variable 3′UTR region is cloned downstream of the CMV promoter and the gfp coding sequence and upstream of a polyadenylation site. For RNA localization measurements (left), the final reporter library is transfected into differentiated neuronal cell lines grown on microporous membranes, and reporter RNA from soma and neurite compartments are collected and sequenced, resulting in a measure for the enrichment of each library variant in the neurite vs. soma compartment. For RNA stability measurements, the reporter library is transfected into differentiated neuronal cell lines, followed by transcription inhibition using Actinomycin D and collection and sequencing of reporter RNA 0, 4 or 24 h later BC: barcode; pA-site: polyadenylation site. ( B ) Density plot of log FC(neurite/soma) measured in CAD (red) and Neuro-2a cells (yellow); n = 47 347 for both. ( C ) log FC(neurite/soma) measured in CAD cells are plotted against the logFC measured for the same sequence in Neuro-2a cells, for all sequences tested (light blue, n = 47 347) or only those with statistically significant enrichment (neurite or soma, P = 0.05) in both cell lines (red, n = 3412). ( D ) Density plot of logFC(4 h/0 h Actinomycin D) (yellow) or logFC(24 h/0 h Actinomycin D) (red) measured in CAD cells; n = 13753 for both. ( E ) logFC(4 h/0 h Actinomycin D) are plotted against logFC(24 h/0 h Actinomycin D) measured for the same sequences in CAD cells, for all sequences tested (light blue, n = 13 753) or only those with statistically significant effect on RNA levels (in 4 h and 24 h, P = 0.05; red, n = 2542). ( F ) logFC(4 h/0 h Actinomycin D) measurements are plotted against logFC (neurite/soma) as determined in CAD cells; library variants with logFC (neurite/soma) <0 are shown in light blue and the corresponding trend line in orange, library variants with logFC(neurite/soma) >0 are shown in dark blue and the corresponding trend line in red ( n = 13 753).

Article Snippet: The master plasmid for inserting the library was created by introducing a synthetic sequence containing a stop codon, a primer binding site and restriction sites for SgsI and SdaI into the Bsp1704I site at the 3′ end of the GFP coding region of pcDNA3-EGFP (Addgene #13013).

Techniques: Reporter Assay, Amplification, Clone Assay, Sequencing, Transfection, Variant Assay, Inhibition