Neuronal zipcode identification protocol (N-zip) in primary cortical neurons

For identification of transcripts enriched in neurites of primary cortical neurons, the cells were plated on a microporous membrane for isolation of neurites and soma. Extracted RNA was used for preparation of mRNA-seq, total RNA-seq or smRNA-seq libraries and Illumina sequencing. For neuronal zipcode identification protocol (N-zip) in mouse cortical neurons, we combined a massively parallel reporter assay with neurites/soma separation. Neurons, grown on a microporous membrane, were infected with a library of around 5000 oligos tiled across 3'UTRs of selected neurite-enriched transcripts, cloned downstream of GFP coding sequence. RNA was extracted from soma and neurites and reverse transcribed into cDNA. Amplicon libraries of 3'UTR reporters were prepared and subjected to Illumina sequencing. In the second round of N-zip, a library containing selected reporters from the first N-zip and their mutagenized versions (around 6000 oligos) were used. In the following rounds, N-zip was combined with knockdown of selected genes.