Bioanalyzer rna6000 pico

Six patient samples were chosen for RNA-seq analyses. The criteria for this selection were: (1) same tissue origin, enough material and possible cut-off between long and short survivals. Total RNA was isolated from tumour cells of formalin fixed paraffin embedded (FFPE) tissue sections using the RNeasy FFPE Kit (Qiagen) according to manufacturer’s instructions. Each sample was processed in duplicates. RNA quality control was performed using the Bioanalyzer RNA 6000 Pico (Agilent). RNA-seq libraries were prepared using SMARTer Universal Low Input RNA Kit for Sequencing (Clonthech) according to manufacturer’s instructions. Sequencing was performed using the Illumina HiSeq 3000 Sequencing System (75 bp paired end sequencing). Raw RNA-seq data were aligned to hg19 using TopHat2 algorithm^{28 (link)}. Differentially expressed transcripts were detected using DESeq2 (version 1.14.1) and EdgeR (version 3.16.5) algorithms^{29 (link), 30 (link)}. The presence of fusion transcripts was analysed using FusionCatcher³¹ .

Total RNA was purified from cells with the InvitrogenTM TRIzolTM Plus RNA Purification Kit. The RNA samples were treated with DNaseI for 1 h at 37 °C. This was followed by the addition of 50 mM EDTA and heat inactivation of the enzyme at 65 °C for 10 min. As a quality control, the RNA was analysed with the Agilent Bioanalyzer RNA 6000 pico assay. All RNA samples used for further processing had an RNA integrity number (RIN) of 7.3 or higher. Reverse transcription was carried out using the Thermo ScientificTM RevertAid First Strand cDNA Synthesis Kit. The cDNA was diluted up to 1:4.

RNA from the CD4⁺ cell cultures was prepared using the Norgen Total micro mRNA kit (Norgen, Ontario, Canada). Quality control was done by Bioanalyzer RNA6000 Pico on Agilent2100 (Agilent, Santa Clara, CA, USA). Deep sequencing was done by RNAseq (Hiseq2000, Illumina) at the Science for Life Laboratory, Huddinge, Sweden. Raw sequence data were obtained in Bcl-files and converted into fastq text format using the bcl2fastq program from Illumina.