title: RNA-seq of human brain: autism temporal cortex: Sample S5
design: Brain samples were dissected retaining gray matter from all cortical layers, isolating 50–100 mg of tissue across the superior temporal gyrus (Brodmann areas ba41/42/22). RNA was isolated using the miRNeasy kit (Qiagen). Ribosomal RNA was depleted from 2ug total RNA with the Ribo-Zero Gold kit (Epicenter). Remaining RNA was then size selected with AMPure XP beads (Beckman Coulter) and resuspended in 8.5 ml of Illumina resuspension buffer and 8.5 ml of 23 EPF buffer. Subsequent steps followed the standard Illumina TruSeq v2 protocol. Libraries were quantified with the Quant-iT PicoGreen assay (Life Technologies) and validated on an Agilent 2200 TapeStation system. These samples, as well as others, were pooled to multiplex 24 samples per lane using Illumina barcodes, and each pool was sequenced six times on a HiSeq2500 instrument using high output mode with standard chemistry and protocols to achieve an average depth of 64M. Throughout dissection, library preparation, and sample pooling, samples were randomized over age, sex, and diagnosis.