RNAi of signal transduction components in HCT116 cells
RNAi of signal transduction components in HCT116 cells
SS000025
HCT116 cells were grown in McCoy's 5a medium (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (PAA, Pasching , Austria) without antibiotics RNAi knockdown in HCT116 cells (ATCC) was performed by reverse transfection in 12 well format.  Ambion silencer select RNAi reagents (Ambion) were used at 10nM final concentration using a 1:1000 final dilution ofDharmafect 1 (Dharmaon). Cells wer seeded at 100000 cells/well in a total volum of 1ml and incubated for 3 days. 2 wells were pooled for each treatment. Total RNA was extracted using the RNAeasy kit (Qiagen, Hilden, Germany). RNA integrity was verified on an Agient 2100 Bioanalyzer. mRNA was isolated with the Dynabeads mRNA DIRECT Kit (Invitrogen, Carlsbad,CA). For each sample, a library for SOLiD sequencing was prepared with the Whole transcriptome analysis kit (4409491 Rev. D, Invitrogen, Carlsbad,CA). Briefly, polyA RNA was fragmented using RNAseIII, purified, hybridized and ligated to RNA adapters. Ligation products were reverse transcribed, purified and ca. 200 nt cDNA fragments selected after electrophoresis on a 6% TBE-Urea gel. cDNA was PCR amplified with SOLiD sequencing primers and purified to produce a library suitable as a template for emulsion PCR. DNA concentrations were determined using qPCR and a pooled library was generated by combining equimolar amounts from all samples.