title: NextSeq 500 sequencing; RNAseq of HCT116 p53KO cells transfected with either siRNA silencing ZNF84 or with negative control siRNA, in 2 variants of experiment a) with doxorubicin treatment (doxorubicin added 48h post-transfection, for the next 48h before harvesting) b) untreated, cultured 72h post-transfection
design: RNAseq of HCT116 p53KO cells transfected with either siRNA silencing ZNF84 or with negative control siRNA, in 2 variants of experiment a) with doxorubicin treatment (doxorubicin added 48h post-transfection, for the next 48h before harvesting) b) untreated, cultured 72h post-transfection
library_construction_protocol: The human colon HCT116 p53KO cells were seeded at a density of 5000 cells per cm2. After 24 hours they were transfected with 30 nM siRNA (against ZNF84 or negative control siRNA) (Thermo Fisher Scientific) using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer's protocol. 24 hours after transfection the medium was replaced with fresh McCoy's suplemented with FBS, in which the cells were cultured for the following 48 hours and then RNA was isolated.  RNA was isolated using RNeasy® Plus Mini Kit (Qiagen). RNA integrity was checked using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies), and concentration was measured with Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies). Stranded mRNA-Seq libraries were prepared from 300ng of total RNA using the Illumina TruSeq RNA Sample Preparation v2 Kit (Illumina) implemented on the liquid handling robot Beckman FXP2. Obtained libraries that passed the QC step, which was assessed on the Agilent Bioanalyzer system, were pooled in equimolar amounts.