DIV-4.1
Embryonic stem cells (ESCs) were maintained and differentiated into highly enriched glutamatergic neurons as described in Hubbard, et al (BMC Neuroscience 2012, 13:127).   High quality RNA was isolated from ESCs (DIV -8; n=4); neuroepithelial stem cells (DIV -4; n=3); radial glia (DIV 0; n=3); developmental stage (DS) I/II neurons (DIV 1; n=4); DS III/IV neurons (DIV 7; n=5); and maturing DS IV/V neurons at DIV 16 (n=4); DIV 21 (n=4); and DIV 28 (n=4) using the QIAcube (Qiagen, Valencia, CA) with the RNeasy mini kit protocol.  PolyA+ RNA was purified from 500 ng of total RNA using polyA selection, chemically fragmented and reverse transcribed using random hexamers.  This was followed by second strand synthesis and end repair.  Libraries were prepared for paired-end sequencing using the TruSeq™ RNA sample prep kits (Illumina, San Diego, CA) per manufacturer’s instructions and library size and integrity were determined using the Agilent Bioanalyzer 2100 (Santa Clara, CA).  Libraries were bound to flow cell surfaces using the Standard Cluster Generation Kit v5 (Illumina), flow cells were transferred to the Illumina HiSeq 2000 and run using TruSeq SBS Kits (Illumina).  Paired-end sequencing data was generated over 50 cycles