RNAi of signal transduction components in Drosophila S2 cells
RNAi of signal transduction components in Drosophila S2 cells
S2 cells adjusted to growth in serum-free media (D.Mel-2, Invitrogen, Carlsbad, CA) were grown in Express Five SFM (Invitrogen, Carlsbad, CA) supplemented with 20 mM Glutamax I, 100 U/ml penicillin, 100 microgram/ml streptomycin. RNAi knockdown was performed by seeding 0.75 Mio cells in 1.5 ml Express-5 medium containing 20 microgram dsRNA and 8 microliter 0.4 mg/ml dimethyldioctadecylammonium bromide (DDAB) per well of a 6-well plate. Cells were incubated at 25 degr. C for 5 days and harvested by centrifugation at 1500xg for 5 min. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA), followed by Rneasy cleanup (Qiagen, Hilden, Germany) including on-column DNase digest. 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.
Experimental Factor: RNAI
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