Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was isolated from SKH-1 cells by TrizolTM (Life Technologies, US) as by manufacturer’s instructions. At the last step of the protocol RNA was resuspended in 17µl of RNAse free water to which was added 2µl of 10x buffer supplied with the Ambion Turbo DNaseI (Thermos Scientific, USA), of which 1 µl was added. All of which was incubated at 37ºC for 30 minutes. The RNA solution was then purified using a Nucleospin RNA clean up column (Machery Nagel, France), according to their instructions. The quality of RNA from all methods was assessed using a spectrophotometer, by the ratio of the absorbance at 260nM and 280nM wavelengths. RNA has a greater absorbance in the 260nM wavelength, Eukaryotic Total RNA PICO Bioanalyser chip (Agilent technologies, USA) allows visualisation of the size of the RNA molecules and thus, demonstrates whether the sample is degraded or not RNA-seq libraries were prepared with a Total RNA Ribo-zero library preparation kit (with ribosomal RNA depletion) (Illumina, USA) according to manufacturer’s instructions with the following alterations: 15 cycles of PCR was undertaken to amplify the library and adaptors for multiplexing were used at a 1:4 dilution. Library quality was checked by running the samples on a Bioanalyser and libraries were quantified using a Kapa library quantification kit (Kapa Biosystems, USA) and run in a pool of eight indexed libraries in two lane of a HiSeq 2500 (Illumina, USA) using rapid run chemistry with 100bp paired end reads.