title: NextSeq 500 sequencing; Stable over expression of the SPOP-substrate ZMYND11 in VCaP cells
library_construction_protocol: For over-expression a derivate of the pLX304 vector was used in which the CMV promoter has been exchanged to a PGK promoter and the blastocidin cassette exchanged by a puromycin resistance cassette (pLX_TRC_307, available at Addgene as Plasmid 41392, pCW107). All ORFs were cloned into pLX_TRC_307-mOrange using Nhe1 and Mlu1. After infection, cells were selected in the presence of puromycin (2 ug/ml). VCaP were grown in DMEM (Gibco) and supplemented with 10% FBS (Gibco), 1% Penicilin/Streptomycin/L-Glutamate. All cells were incubated at 37°C and 5% CO2. Cells were routinely tested for mycoplasma contamination. RNA was extracted using the Rnasy kit (Qiagen) and sample quality was assessed using Agilent Bioanalyzer. RNA libraries were prepared for sequencing using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®