title: GSM4259964: Tet-Twister-screen1-32, 50 uM Tetracycline, rep 8; Homo sapiens; OTHER
library_construction_protocol: RNA was extracted using the Qiagen RNeasy 96 kit. Following random priming-based reverse transcription to cDNA, sequence amplicons of interest were generated by non-saturating PCR and PCR amplicons were purified. DNA concentration was determined on the Fragment Analyzer with NGS StandardSensitivity according to manufacturer's instructions. Subsequently, end repair was carried out using the Ovation Library System for Low Complexity Samples kit (NuGEN) with the manufacturer's protocol, followed by adaptor ligation from the same kit. The libraries underwent cleanup again using the MagMAX Express-96 Magnetic Particle processor and the Agencourt RNAClean XP beads (Beckmann Coulter) following the manufacturer's protocol. Subsequently, library amplification was carried out using the Ovation Library System for Low Complexity Samples kit (NuGEN) with manufacturer's protocol, followed by a clean-up step with Agencourt AMPure XP Beads (Beckmann Coulter). The libraries were quantified with a Quant-iT PicoGreen dsDNA Assay kit (Invitrogen) according to manufacturer's instructions and fragment size was determined with NGS Standard Sensitivity Kit (Advanced Analytical) on a Fragment Analyzer. Based on the concentrations obtained from the PicoGreen measurement, a 2 nM dilution of the libraries in Illumina Resuspension Buffer + 0.1% Tween20 preparation (Sigma) was prepared and in the following 10 µl of each sample were mixed with a unique reverse adaptor from the 2 nM dilution. Libraries were denatured by consecutive addition of 10 µl 0.2 N NaOH, µl of 200 mM Tris-HCl, pH 7.0 and 970 µl of prechilled HT1 buffer. In a new prechilled tube, 1183 µl of prechilled HT1 buffer were added. Subsequently 117 µl of the library pool dilution and 2 µl of 20 pM PhiX control were added. Finally, the libraries were sequenced on an Illumina NextSeq 500, using 10 million single-end reads of 154 bp length  per sample, using the NextSeq 500/550 High Output Kit v2.
attributes.tag: GEO Accession
attributes.value: GSM4259964