GSM5912842: PTEN knock-out normoxia rep2; Mus musculus; miRNA-Seq
Total RNA was extracted from cancer cells cultured in normoxia and hypoxia or tumor tissues using RNeasy Mini Kit (Qiagen, Germany). The samples were freed from DNA using TURBO DNA-free kit (Thermo Fisher Scientific, USA) and RNA quality and concentrations were evaluated using the fluorometer Qubit (Qubit RNA BR Assay Kit, RNA with Qubit RNA IQ Assay Kit, Thermo Fisher Scientific, USA) according to manufacturers' instructions. Total RNA from Renca cells and tumors (pten/WT, pten/KO) was used to prepare libraries and perform high-throughput sequencing by an external service Lexogen GmbH (Vienna, Austria) with NextSeq 500 system (Illumina, USA). Briefly, RNA concentration was measured by UV-Vis spectrophotometry (Nanodrop 2000c, Thermo Fisher), and RNA integrity was assessed on a Fragment Analyzer System using the DNF-471 RNA Kit (15 nt) (Agilent, USA). Sequencing-ready libraries were produced using a Small RNA-SeqLibrary Prep Kit by Lexogen (052UG128V0110) per manufacturer instructions. Indexed library preparation was performed to allow for multiplexed sequencing. For library preparation, 100ng of each provided RNA sample was used. All libraries were analyzed for adapter dimers, size distribution and concentration on a Fragment Analyzer System using the DNF-474 HS NGS Fragment kit (1-6,000bp) (Agilent, USA). Libraries were pooled in an  equimolar ratio. After pooling, 1,3x magnetic bead purification and agarose gel size selection was performed to target miRNA fraction. The concentration and the size distribution of the final lane mix was analyzed by Qubit dsDNA HS assay (Thermo Fisher Scientific) and by Fragment Analyzer System using the DNF-474 HS NGS Fragment kit (1-6,000bp) (Agilent, USA). A 2 nM dilution of the lane mix was denatured and diluted to loading  concentration for sequencing on a NextSeq 500 instrument with SR75 High Output Kit (Illumina).