Pooled Evrogen SMART cDNA normalized samples of various treatments of Anastatica hierochuntica
Tissue was snap-frozen in liquid nitrogen, and 100 mg of tissue was ground using a pestle and mortar, followed by RNA isolation using the RNeasy Mini Kit (Qiagen, 2010). RNA purity and concentration were determined by NanoDrop spectrophotometer (NanoDrop ND-1000) and by formaldehyde agarose gel electrophoresis. The quality of RNA was further evaluated on an Agilent 2100 Bioanalyzer (Agilent). Equal amounts of total RNA were pooled together, and sent in GenTegra tubes for cDNA library construction and sequencing. To reduce abundant RNAs such as Rubisco, and therefore enable detection of rare RNAs, a normalized cDNA library was constructed using the Evrogen SMART technology cDNA synthesis service (Evrogen, Moscow, Russia), and was sequenced with a full picotiter plate on a 454 GS-FLX sequencer with Titanium reagents (Roche Applied Science, Indianapolis, IN, USA), in the GenePool genomics facility at the University of Edinburgh, UK.