RNA-seq of HT1080 clone 5C3 with knocked out HSP90AA1 gene, replicate 3
Total RNA was prepared from parental HT1080 cells and from Hsp90-deficient 5C3/7D5 and 2C8/7F10 cell clones using a Total RNA Purification Kit (Jena Bioscience, Jena, Thuringen, Germany). For each cell line, the RNA was prepared from cell samples grown in three independent cell culture flasks to a 6080% confluence. The RNA concentration was determined with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, MA, USA). The total RNA quality was determined using agarose gel electrophoresis. Purified total RNA was stored at -800C. To eliminate genome DNA impurities, total RNA was treated with recombinant duplex-specific nuclease (Evrogen, Moscow, Russia). The depletion of ribosomal RNA and the construction of sequencing libraries were performed using a TruSeq Stranded Total RNA Human Kit (Illumina, CA, USA) according to the manufacturers protocol. The quality of the libraries was evaluated with a Fragment Analyzer (Agilent Technologies, CA, USA).