title: Illumina HiSeq 2000 sequencing
library_construction_protocol: Strand-specific RNA-seq libraries were prepared from poly(A)-enriched (poly(A)+) RNA using an unreleased early version of the Illumina TruSeq Small RNA Sample Prep Kit.  Briefly, 10mg of total RNA was used as starting material and poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic bead purification.  RNA was fragmented to an average size of ~200 nucleotides (nt).  Fragmented RNA was 3_ de-phosphorylated with Antarctic phosphatase and 5_ phosphorylated with polynucleotide kinase; this treatment prepared RNA fragments for subsequent ligation of Illumina RNA adaptors to their 5_ and 3_ ends using a 3_ RNA ligase and a T4 RNA ligase respectively.  First-strand cDNA was produced using a primer specific for the Illumina 3_ adaptor.  The library was amplified with 15 PCR cycles using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter).  Library size distributions and concentrations were determined on a Bioanalyser (Agilent).