Neural cells were fluorescently labeled using GFP under the control of ase regulatory sequences and FACS-sorted into neuroblasts and neurons. Neuroblasts were distinguished by their strong GFP signal, large size and relatively small population, while neurons were identified by their weaker GFP signal, smaller size and greater abundance.
Poly(A)+ RNA was subjected to first strand cDNA synthesis. Second strand cDNA synthesis was done in the presence of UTP to generate stranded cDNA libraries. Double-stranded cDNA was end-repaired and ligated to adapters. After size selection (200-600bp) and UDG-ase treatment for strand specificity, DNA was PCR amplified and the quality assessed by Agilent bioanalyzer.
Total RNA was isolated from 28-35 million FACS-sorted neurons using Trizol. Polyadenylated mRNA was enriched for using oligo(dT) beads. The poly(A)+ RNA was fragmented using divalent cations under elevated temperature.
Read length (bases):76