GSM4720861: 23_T23_FIBROB6_ENZA_ISO_S7; Mus musculus; RNA-Seq
Total RNA was isolated from cell lines by means of miRNeasy Tissue/Cells Advanced Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA quality and quantity were checked using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, U.S.) and Nanodrop spectrophotometer (Thermo Fisher, Waltham, Massachusetts, U.S.). 500 ng of totRNA were used to prepare RNA libraries by means of the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, California, U.S.) following the manufacturer's protocol. Briefly, the mRNA fraction was purified from total RNA by polyA capture, fragmented and subjected to first-strand cDNA synthesis with random primers in the presence of Actinomycin D. The second-strand synthesis was performed incorporating dUTP instead of dTTP. Then a single 'A' nucleotide was added to the 3' ends of the blunt fragments to prevent them to ligating to each other. Index adapters were ligated and cDNA was subjected to PCR amplification. Libraries were validated using Agilent bioanalyzer (DNA 1000 kit) and the concentration was evaluated by means of Qubit™ dsDNA HS Assay Kit (Thermo Fisher). Libraries were normalized, multiplexed and sequenced on the Illumina NextSeq 550 system.
GEO
GSM4720861