title: RNA-seq of Pan troglodytes: adult mediodorsal nucleus of the thalamus
design: cDNA libraries were prepared using the mRNA-Seq Sample Kit (Illumina) as per the manufacturer's instructions with some modifications. Briefly, polyA RNA was purified from 1 to 5 µg of total RNA using (dT) beads. Quaint-IT RiboGreen RNA Assay Kit (Invitrogen) was used to quantitate purified mRNA with the NanoDrop 3300. Following mRNA quantitation, 2.5 µl spike-in master mixes, containing five different types of RNA molecules at varying amount (2.5 × 10-7 to 2.5 × 10-14 mol), were added per 100 ng of mRNA. The spike-in RNAs were synthesized by External RNA Control Consortium (ERCC) by in vitro transcription of de novo DNA sequences or of DNA derived from the B. subtilis or the deep-sea vent microbe M. jannaschii genomes and were a generous gift of Dr. Mark Salit at The National Institute of Standards and Technology (NIST). These were used both to track the brain regions and to normalize expression levels across experiments. Each sample was tagged by adding a pair of spike-in RNAs unique to the region from which the sample was taken. Also, an additional three common spike-ins were added for controlling sequencing error rates, which is not influenced by SNP existence. The mixture of mRNA and spike-in RNAs were subjected to fragmentation, reverse transcription, end repair, 3'-end adenylation, and adapter ligation to generate libraries of short cDNA molecules. The libraries were size selected at 200-250 bp by gel excision, followed by PCR amplification and column purification.