A Multi-Faceted Analysis Showing CRNDE Transcripts and a Recently Confirmed Micropeptide as Important Players in Ovarian Carcinogenesis
For two independently developed SK-OV-3 cell lines with stable silencing of CRNDE with the SH1 shRNA (SK-OV-3-SH1-K1, SK-OV-3-SH1-K2) and two corresponding control cell lines (SK-OV-3-SCR-K1, SK-OV-3-SCR-K3), a whole transcriptome evaluation was carried out by NGS RNA-seq in three biological replicates, giving 12 samples in total. This study involved total RNA extraction (from 1 million cells per sample) with the Pure Link RNA mini kit (Thermo). Then, RNA was treated with the Turbo DNA-free Kit (Thermo) to remove all traces of DNA from the samples. RNA concentrations were assessed on the Quantus Fluorometer, using the QuantiFluor RNA System kit (Promega, Madison, WI, USA). RNA quality evaluation by RNA integrity number (RIN) estimation (all the RINs = 9) was carried out with the Agilent RNA 6000 Nano Kit on the 2100 Bioanalyzer (both manufactured by Agilent Technologies, Santa Clara, CA, USA). Next, 1 µg of total RNA was reverse transcribed to cDNA with the SuperScript II kit (Thermo). The obtained cDNA was then used to generate NGS libraries with the TruSeq Stranded Total RNA Library Prep Gold (Illumina, San Diego, CA, USA), according to the recommendations of the kit's manufacturer. The VAHTS RNA Clean Beads (Vazyme Biotech, Nanjing Shi, China) were applied to clean cDNA after every enzymatic reaction. Subsequently, the distribution of molecules' lengths in the libraries, as well as the cDNA concentration in each library were assessed on the 2100 Bioanalyzer by using the Agilent High Sensitivity DNA Kit (Agilent Technologies). Finally, all the libraries were pooled in equimolar ratios to create the multi-library, additionally supplemented with the PhiX Control v3 Library (Illumina) in the final conc. of 1%, and then sequenced on the Illumina NovaSeq 6000 platform in the paired-end mode (2x100 bp). The resultant NGS RNA-Seq FASTQ files have been submitted to the European Nucleotide Archive (ENA) database (data acc. no. PRJEB61091).