NextSeq 500 sequencing; mRNA-seq of neuronal compartments with perturbed mRNA degradation machinery in mouse primary cortical neurons (mPCN)
mRNA-seq of neuronal compartments with perturbed mRNA degradation machinery in mouse primary cortical neurons (mPCN)
Primary neuron cultures were prepared from C57B/6 wild type mice (P0) pups and cultured as previously described (Kaech and Banker, 2006). RNA was extracted from Trifast lysate using Directzol column purification kit (zymo) including DNaseI treatment procedure; RNA samples were quantified and analysed for integrity by Bioanalyser; Good separation was verified by RT-qPCR. (2.5-3.7 cycles difference in Taglin between soma/neurite samples) Stranded mRNA Library kit; 1ul of 1:100 ERCC1 spike-in was added to 49ul initial solution with 400ng total RNA.; Final PCR was done with 10-15 cyclesâ€™ reaction.