title: 454 Rhazya Stricta transcriptomics
design: All material was collected from a site at Bahrah near Jeddah in the Kingdom of Saudi Arabia Leaves were picked, wrapped in aluminium foil, labelled and snap frozen in liquid nitrogen and then stored at -80 °C until required for extraction.     Leaf tissue was ground to a fine powder in liquid nitrogen using a pestle and mortar, then approx 100mg of tissue was RNA extracted using Trizol® (Life technologies, Carlsbad, USA) as described by the manufacturer. The RNA was then purified using a Spin Column Reaction Cleanup Kit (NBS Biologicals, Huntingdon, UK). The RNA was then purified using a Spin Column Reaction Cleanup Kit (NBS Biologicals) as described by the manufacturer.    16 µg total RNA was sent to Evrogen Ltd (Moscow, Russia) for library normalisation using service CS010-1A (http://www.evrogen.com/services/cDNA-normalization/service-cdna-normalization_Terms2.shtml) Briefly, total RNA sample was used for double-stranded cDNA synthesis using the SMART approach (Zhu et al, 2001). SMART-prepared amplified cDNA was then normalized using the duplex-specific nuclease (DSN) normalization method (Zhulidov et al, 2004). Normalization included cDNA denaturation/re-association treatment (DSN, Shagin et al, 2002) and amplification of the normalized fraction by PCR.    The normalised library prepared by Evrogen was sent to the Centre for Genomic Research (CGR) (University of Liverpool, UK) for sequencing using 454 GS FLX platform (Roche, Basel, Switzerland). Data were quality checked using FastQC, and quality trimmed using clean_reads (http://bioinf.comav.upv.es/clean_reads), all default settings. All bacterial reads were removed by aligning against all bacterial genomes downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/). Finally the SMART vector was removed using fastx_clipper (http://hannonlab.cshl.edu/fastx_toolkit/index.html)