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RNA sequencing (RNA-Seq) is a next-generation sequencing method, routinely used in laboratories to detect abnormal transcripts with oncogenic activity after RNA extraction from formalin-fixed-paraffin-embedded (FFPE) samples of non-small-cell-lung cancers (NSCLC), then allowing targeting of the corresponding fusion kinase with adapted pharmacologic compounds in the clinical practice. Here, we provide a detailed protocol for total nucleic acid (i.e., RNA and DNA) extraction from FFPE samples and targeted RNA-Seq, by the FusionPlex^® method which allows the detection of the most current fusion transcripts found in NSCLC.