title: 1024_PFC
design: or  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.  Typical  yields  from  both  non-ASD and Autism subjects were about 10–12 μg of total RNA from 100 mg of tissue. 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.