7 protocols
The Illumina microarray data were analyzed using the R statistics package, specifically using the packages lumi and limma.
Labeled cDNA samples were purified and hybridized to Illumina Human HT-12_V4 expression bead arrays according to the supplier's protocol.
200ng of RNA were amplified and labeled using the Illumina Total Prep 96 RNA Amplification kit from Applied Biosystems.
Slides were washed and scanned using the Illumina iScan BeadStation and signals were quantified and analyzed using the Beadstudio Software from Illumina.
Antibiotic selection using geneticin (200ug/ul) was started after 24 hours. Several clones were picked, expanded in selective medium and checked for DYX1C1 expression. Plasmid and siRNA oligo transfection were made using Lipofectamine 2000 and Dharmafect I, respectively.
Total cellular RNA from the samples was extracted using Rneasy minikit (Qiagen, Hilden, Germany) according to manufacturer's instructions.
For the all the experiments, cells were cultured 24-48 h in phenol-red free MEM, supplemented with L-glutamine and 4% of DCC-treaded FBS. For the generation of DYX1C1 stable SH-SY5Y cell line, DYX1C1 plasmid was linearized using HindIII and EcoRI restriction enzymes and transfected into SH-SY5Y cell line.