title: NextSeq 500 sequencing; Identification of ZMYND11 binding sites in VCaP cells with stable overexpression of either mutant (Y87C) or wild-type SPOP
design: Identification of ZMYND11 binding sites in VCaP cells with stable overexpression of either mutant (Y87C) or wild-type SPOP
library_construction_protocol: For SPOP mutant 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 62511, pCW107). All ORFs were cloned into pLX_TRC_307-mOrange using Nhe1 and Mlu1. ChIP was performed using the anti-ZMYND11 antibody (NBP2-20960, Novus Biologicals) 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. DNA was extracted using the Quiagen kit and sample quality was assessed using Agilent Bioanalyzer. DNA libraries were prepared for sequencing using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®