title: Secretome analysis of human oligodendrocytes derived from neural stem cells
design: Total RNA was extracted using the TRIzol-based method (Life Technologies). After fractionation of total RNA, samples were precipitated with 0.5 M ammonium acetate, and residual salt was removed. The quality of total RNA samples was determined using an Agilent Bioanalyzer, and the samples with the greatest RNA integrity number score (score: 10) were selected for the next step. Enrichment of mRNA was performed using the oligo-dT cellulose method (Poly(A) Purist kit; Life Technologies). Isolated mRNA samples were assessed with the Bioanalyzer to confirm the enrichment of mRNA. mRNA-Seq library samples were prepared using a SOLiD Total RNA-Seq kit (Life Technologies). The amount of starting RNA material for RNA fragmentation was 500 ng. RNA samples were fragmented with RNase III, hybridized, and ligated to SOLiD adapters and ligation enzymes. cDNA was synthesized using ArrayScript™ Reverse Transcriptase and size-selected by separation on a TBE-urea gel. After amplification of cDNA, the yield and size distribution of the amplified cDNA were assessed using the Bioanalyzer.