Illumina NovaSeq 6000 paired end sequencing; RNA-seq of vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia and age-matched and sex-matched healthy controls
Vastus lateralis muscle biopsies of newly diagnosed treatment‐naïve NSCLC patients with cachexia (n = 8) and age‐matched and sex‐matched healthy controls (n = 8) were processed for RNA sequencing. Muscles were grinded into powder and homogenized in TRI Reagent by using a Mini bead beater (Cole Parmer) sample homogenizer, and total RNA was extracted. For mRNA expression analysis, TRI Reagent (Sigma-Aldrich) was used according to the manufacturers' protocol. Pure, intact mRNA was isolated from 1 µg of total RNA using the NEXTFLEX Poly (A) Beads 2.0 kit (PerkinElmer). The purified mRNA was used to prepare directional, strand specific RNA libraries for sequencing using the NEXTFLEX Rapid Directional RNA-Seq kit 2.0 (PerkinElmer) according to manufacturer's instruction. The NETFLEX RNA-Seq 2.0 Unique Dual Index Barcodes (PerkinElmer) were used as adapter. A Qubit fluorometer (Invitrogen) and the Agilent 4200 Tapestation system (Agilent Technologies) were used to measure the quantity and quality of the libraries. Equal amounts (1.6 nM) of libraries were pooled and sequenced using the S2 flow cell (200 cycles) of the Illumina NovaSeq 6000.