RNA-Seq of camel Camelus dromedarius supraoptic nucleus
Tissue was stored in RNAlater at -80OC until extraction in a Category II fume hood. Samples were then weighed and dissected to ensure that each was less than the 30mg to ensure that extraction by column was maximally efficient. Each sample was then homogenized in 1ml Trizol and the aqueous phase separated with Chloroform. Purification relied on the RNeasyMiniKit (Qiagen) according to manufacturers protocol. Concentration and quality of RNA was determined using both Nano-drop and Agilent Bioanalyzer 2100 and samples with a R.I.N>8 were used for library construction. Stranded total RNA libraries were prepared according to IlluminaTruSeq stranded total RNA sample preparation guide (April 2013, Rev D) for Illumina Paired-End Multiplex Sequencing (Source Bioscience, Nottingham, UK). Ribo-Zero gold kit was used to remove cytoplasmic and mitochondrial rRNA prior to fragmentation and priming for first and second strand cDNA synthesis. The 3 end of fragments were adenylated and adapters and indexes (for the multiplex barcoding) ligated. Libraries were then subject to further quality control prior to pools with a concentration of 8pM being loaded onto lanes of an Illumina flowcell and sequenced using 100bp Paired End runs. Each sample resulted in >35million reads.