RNA sequencing of prefrontal cortex samples of autism patients and controls
For RNA extraction ~100 mg of brain tissue was lysed in trizol (Life Technologies), 200 μL of chloroform were added and the sample was incubated at room temperature for 10 minutes. The samples were then centrifuged for 20 minutes at 4°C. The supernatant (aqueous phase) was then transferred to a new tube containing 1.5 volumes of 100% ethanol. The ethanol/RNA mixture was then loaded onto a RNeasy column (Qiagen) and purified as per the manufacturer’s instructions, including on-column DNase treatment. 1 μg of total RNA was then used to prepare directional RNA-seq libraries using NEBNext Ultra Directional Library Prep Kit (New England Biolabs, E7420). After heat-induced RNA fragmentation, cDNA synthesis and adapter ligation the fragments were size-selected using a two-step protocol with AMPure beads (Beckman Coulter, A63880). First,  60 μL of AMPure Beads was added to the 100 μL of the reaction volume contacting cDNA fragments with the adapters, and the beads were with the bound unwanted fragments were discarded. Then, 25 μL of beads was added to the reaction, and the supernatant was discarded. This size selection protocol resulted in a library with an average size of the fragments of 300 bp.
University of Miami
Autis_PFC_RNA-seq