GSM5627761: DU145 A6 clone-empty vector control 3; Homo sapiens; RNA-Seq
RNA was isolated from DU145 A6, D7, B9 and C12 clones (1x10^6 cells) by the use of High Pure RNA isolation kit (cat.n. 11828665001, Roche), which consists of cell lysis, RNA binding to filter, incubation with DNase, washing steps and elution. RNA concentration was evaluated immediately upon isolation by Eppendorf BioSpectrometer. RNA was diluted for each sample at the final concentration 100 ng/ul: 10 ul were transferred to 1.5 ml tube for RNAseq analysis; 500 nanograms of total RNA per sample was used as an input for library preparation using QuantSeq FWD 3'mRNA Library Prep Kit (Lexogen). Briefly, RNA was transcribed into cDNA using oligodT primer (FS1) at half volume compared to the manufacturer's instructions to minimize the off-target products. Following the first strand synthesis, RNA removal and second strand synthesis were performed using the UMI Second Strand Synthesis Mix (USS) containing Unique Molecular Identifiers (UMIs) that allow detection and removal of PCR duplicates. Finally, sequencing libraries were created by PCR with i5 Unique Dual Indexing Add-on Kit for Illumina (Lexogen). The quality and quantity of libraries was determined using Fragment Analyzer by DNF-474 High Sensitivity NGS Fragment Analysis Kit (Agilent Technologies) and QuantiFluor dsDNA System (Promega). Final library pool was sequenced on Illumina NextSeq 500 with 75 bp single-ends