|The protocol is optimized for 0.1–1 µg of total RNA.
Normal and abnormal H9 cells were analysed with mRNA-seq. The RNA was extracted simultaneously from the same cells as DNA with Qiagen Allprep kit. The Next-Generation Sequencing libraries were prepared with Illumina TruSeq mRNA-seq kit according to the protocol from the manufacturer. The libraries were sequenced with 1 × 50 bp chemistry with Illumina HiSeq2000 platform. The total number of reads obtained from sequencing was 19M–28M per sample. The data was analysed in Illumina’s BaseSpace cloud (https://basespace.illlumina.com) with RNA Express v1.0, which applies STAR aligner for read alignment21. Of the total reads 97.51–98.6% were aligned to hg19 reference genome. The differential gene expression analysis, with cut-offs indicated in the results, was carried out in with RNA Express v1.0 and DESeq221,22. Ingenuity Pathway Analysis Tool (Qiagen) was utilized for exploring functions and networks of the altered genes. Full paper
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