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.
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.
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.
Read length (bases):76
Strand-specific paired end reads were filtered for rRNA sequences. Filtered reads were then aligned using TopHat (v1.4.1) against the D. melanogaster genome (FlyBase r5.44) and a maximum of six mismatches. Introns between 20 and 150,000 bp were allowed. snRNA, rRNA, tRNA, snoRNA and pseudogenes were masked.
The RNA-seq profiles displayed by FlyBase in GBrowse and used for RPKM calculation can be accessed at the FTP link below as .wig files. Please take note of how these FlyBase .wig files represent data for a contiguous sequence of bases with the same signal value. The value is declared only for the first position of that region, and applies to all positions that follow (these are not explicitly listed) until a new value at a new base position is declared.