title: Differentiation of human neural progenitor cells using RNA Seq
design: The human neural progenitor cell lines, SK-N-SH and ReNcell CX, were used to measure gene expression as they differentiate into cortical projection neurons.  Cells were transfected with 2 μg of a pIRES2-AcGFP empty vector (Clontech; Mountain View, CA, USA) using Amaxa Nucleofector technology (Lonza; Basel, Switzerland) according to the manufacturer’s protocol.  The cells were then allowed to replicate and undergo differentiation until being harvested either 24 h or 72 h following transfection with the empty vector.  Harvested cells were homogenized with QIAshredder spin columns (Qiagen; Valencia, CA, USA).  Total cellular RNA was extracted using Qiagen RNeasy kit according to manufacturer’s instructions.  RNA quality was assessed using Agilent Technologies 2200 TapeStation Instrument (Agilent Technologies; Santa Clara, CA, USA) and 260/280 absorbance ratios were captured with a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA).  cDNA was made using the Illumina Truseq Stranded Total RNA Sample Preparation kit per the manufacturer’s instructions and Ribozero (Illumina; San Diego, CA, USA) was used to deplete rRNA from the sample.  Libraries were multiplexed using the Illumina Truseq Stranded Total RNA Sample Preparation kit with four samples per lane, pooled and sequenced using the HiSeq2000 sequencer to generate 101 bp single-end reads (Ilumina; San Diego, CA, USA).