title: Illumina HiSeq 4000 paired end sequencing; RNA-Seq of control, RNPS1 knockdown and rescue in HeLa cells
design: RNA-Seq of control, RNPS1 knockdown and rescue in HeLa cells
library_construction_protocol: HeLa Flp-In T-REx cells were cultured in DMEM supplemented with 9% fetal bovine serum and 1x Penicillin Streptomycin. The cells were cultivated at 37°C and 5% CO2 in a humidified incubator. For siRNA transfection, cells were reverse transfected with 60 pmol siRNA targeting RNPS1 (or Luc in the negative control) per 2 x 10^5 cells using 2.5 μl Lipofectamine RNAiMAX. For rescue, for generating stable RNPS1 rescue cell lines, the expression constructs were cloned into the pcDNA5/FRT/TO vector containing an N-terminal FLAG-tag. Standard protocols were used to generate stable rescue or reporter HeLa Flp-In T-REx cell lines and positive clones were selected with 100-150 μg/ml hygromycin B. Expression of stable cell lines was induced with 1 μg/ml doxycycline 24 h after siRNA transfection. Fresh medium with 1 μg/ml doxycycline was added after additional 48 h. The H176D-E179R mutation of a surface exposed patch of RNPS1 prevents ASAP complex formation. Total RNA was extracted with peqGOLD TriFast according to the manufacturer's instructions.  Ribosomal depletion and strand specific library preparation was carried out with the TruSeq R Stranded Total RNA LT (with Ribo-ZeroTM Human/Mouse/Rat) according to the manufacturer's instructions.
attributes.tag: Experimental Factor: phenotype
attributes.value: wild type phenotype