RNA from 4 wells per neural culture was pooled as input for Next Generation Sequencing (NGS). RINs ranged from 7–9.7, with an average of 8.63. Poly-A selected (n=5) or ribosomal-RNA depleted (n=11) RNA was used to generate randomly primed libraries (200–500 bp inserts). NGS was conducted at the Genomics Core of the Yale Stem Cell Center using Illumina TruSeq Chemistry for library preparation and the Illumina HiSeq 2000 platform to generate 100-bp reads (average 44 million reads per sample). Sequence reads were aligned using Bowtie and TopHat (Trapnell et al., 2009) and gene expression levels quantified using Cufflinks (Trapnell et al., 2010) using a computational pipeline in the Department of Computer Science and Engineering, UConn-Storrs. Sequence tag reads were aligned to reference human genome hg19 and results normalized for exon length and the total sequence read number for each sample to generate Reads Per Kilobase of exon per Million mapped reads (RPKM).
