title: Transcriptome-driven constraint-based modelling reveals metabolic targets for ovarian cancer
design: Transcriptome-driven constraint-based modelling reveals metabolic targets for ovarian cancer
library_construction_protocol: OV56 were cells harvested from 96-well plates, after 48-hour incubation with siRNA for test conditions.  OV56 cells (1,300 cell/well) in a 96-well plate. Cultured in 1:1 ratio of DMEM:F12 with 5% FBS, 2mM glutamine, 0.5ug/mL hydrocortisone, 10ug/mL insulin and 50ug/mL penicillin/streptomycin.  Transfection with ON-TARGETplus SMARTpool Human TPI1 siRNA or NTsiRNA. Negative controls treated with transfection reagent only or untreated. Final concentration of 66nM siRNA in a seeding density of 1300 cells/well. Incubation for 48 hours.  RNA was isolated from harvested cells using the RNeasy mini kit.  Poly-adenylated mRNA extracted from total RNA using poly-T, oligo-attached magnetic beads. mRNA fragmented under elevated temperature and reverse transcribed into first strand cDNA using random hexamer primers and Actinomycin D. Second strand cDNA synthesised to yield blunt-ended, double-stranded cDNA fragments. Strand specificity maintained by dUTP-incorporation. Following a single adenine base addition, adapters with a corresponding, complementary thymine overhang were ligated to the cDNA fragments to prepare for dual indexing. PCR amplification used to add the index adaptor sequences to create final cDNA library. 