Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Pelleted cells from primary patient material were lysed by adding 1ml Trizol ™ (Life Technologies, US). 200µl of chloroform was added and the mixture was manually shaken for 15 seconds. The mixture was incubated at room temperature for 3 minutes. The mixture was centrifuged at 12000 x g for 15 minutes at 4°C. The top clear aqueous phase was removed and placed in a fresh tube. 0.5ml of 100% isopropanol was added to the isolated aqueous phase and incubated at room temperature for 10 minutes whichs was then transferred to a RNeasy MinElute column (Qiagen, USA) and centrifuged for 15s at 8000xg. 350µl of RWI buffer from the RNeasy Kit was added to the column and centrifuged for 15s at 8000xg. DNaseI 10µl and 70µl RDD buffer (Qiagen, USA) were mixed and added to the column and incubated for 15 minutes at room temperature. Afterwards, 350µl of RWI buffer from RNeasy Kit (Qiagen, USA) was added to the column and centrifuged for 15s at 8000xg. Following this 500µl of RPE buffer was added and centrifuged for 15s at 8000xg. The column was washed with 500µl 80% ethanol and centrifuged at 2 minutes at 8000xg. The column was dried by centrifuged at 5 minutes at 8000xg. RNA was eluted from the column by adding 12µl of water to the column followed by centrifugation at 5 minutes at 8000xg. 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.