title: NextSeq 500 sequencing; Neuronal zipcode identification protocol (N-zip) in primary cortical neurons
design: Neuronal zipcode identification protocol (N-zip) in primary cortical neurons
library_name: PCN_mRNA_shScramble-Ago_Neurite_3_s
library_construction_protocol: Soma (neuronal cell body) samples were collected from top of the filter membrane and neurite (neuron projection) samples frpm 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. 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 manufacturers instructions (see also Ludwik et al.  2019). 100 ng of total RNA from each sample were used for libraries preparation with either TruSeq stranded mRNA library preparation kit (Illumina 2002059),  Truseq Stranded Total RNA Library kit (Illumina, 15032619) or SMART smRNaseq kit (Takara, 635029) according to the manufacturers recommendations.