title: Illumina NovaSeq 6000 paired end sequencing; Neuronal zipcode identification protocol (N-zip) in primary cortical neurons
design: Neuronal zipcode identification protocol (N-zip) in primary cortical neurons
library_construction_protocol: Soma samples were collected from top of the filter membrane and neurite samples form the cleaned membranes at DIV9. See also: “Ludwik et al.  2019: Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons” Cortical neurons were isolated from E14 and P0 pups and plated on microporous membranes in 6-well plates. Astrocytes were pre-seeded on the 6-well dishes around one week in advance of plating. Cells were cultured as previously described (Kaech & Banker, 2006). Neurons were transfected using lentiviral particles carrying either shRNA expression constructs or GFP constructs with inserts from one of the reporter library pools or. Viral particles were applied onto the neurons between DIV1 and DIV5. After collection samples were immediately transferred into Trizol and RNA extraction was performed according to manufacturer's instructions (see also Ludwik et al.  2019). 100-200ng of total RNA was reverse transcribed into cDNA with a primer complementary to the 3' adapter flanking the tiles. UMIs were introduced during the second strand synthesis with a pool of primers complementary to the 5' adapter flanking the tiles.