title: Koala buffy coat (ISIS # 510113) transcriptome raw and processed reads
design: During routine health exams, whole blood was extracted from two individual koalas (ISIS #: 504219 and 510113).  Buffy coat was extracted from the blood samples via Pasteur pipet after centrifugation for 20 min at 2000 rpm (~900x g).  The buffy coat was then stored in TRIzol and frozen.  The sample was shipped overnight on dry ice to Purdue University, where it was stored at -80 degrees Celsius.  RNA was extracted using TRIzol (Life Technologies) and Direct-zol columns (Zymo Research) via manufacturer's instructions.  Approximately 5 ug of RNA was used to create cDNA libraries using random hexamer primers according to the TruSeq RNA sample preparation manual (Illumina).  The libraries were barcoded, pooled, and then sequenced on a lane of the Illumina HiSeq 2000 platform.  The "raw" reads are those resulting from the previous steps.  The "processed" were the result of further filtering.  Specifically, the adaptor sequences were removed, the reads were filtered for quality (PHRED quality score > 20 and length > 30 bp), and putative ribosomal RNA reads were removed using DeconSeq and a custom vertebrate rRNA database.