array scanning protocol
GeneChips were scanned using Affymetrix GeneChip 3000 7G
normalization data transformation protocol
The data were background corrected and normalized using the robust multiarray average (RMA) procedure implemented in the Affymetrix Expression Console software. probe group file: MoEx-1_0-st-v1.r2.pgf meta-probeset file: MoEx-1_0-st-v1.r2.dtl.mm9.full.ps ID_REF = VALUE = Quantification DETECTION P-VALUE =
Samples were hybridized with GeneChip mouse exon 1.0 ST Arrays (Affymetrix) and scanned accoding to the manufacture's protocol.
100ng of total RNA were used to generate labeled cDNA. Biotinylated DNA probes were prepared using Affymetrix WT Expression kit for Affymetrix Whole Transcript Sense Expression arrays and Affymetrix geneChip WT Terminal Labeling kit according to the manufacture's protocol.
sample treatment protocol
On day 5, neurons were infected with 2 x 1010 copies/well (1.5 x 107 copies/µl) of lentivirus expressing shRNA against mouse Cugbp1 (shCugbp1) or control (shCont). After 4 hr of infection, the virus media was removed. Neurons were then cultured for 6 additional days, and were harvested on day 11 followed by RNA extraction and cDNA synthesis.
Mouse fetal brain was taken from C57BL/6 mouse embryos at E15. After removing meninges cortical tissue was dissociated into a single-cell suspension by Sumilon dissociation solution (Sumitomo Bakelite, Tokyo Japan). Cells were plated at a density of 1.5 x 106 cells in a 60 mm-well culture plate with the medium containing 0.5x Sumilon nerve-culture medium (Sumitomo Bakelite), 0.5x Neurobasal medium, 1% FBS, 0.5x B27 supplements (Invitrogen), 0.5x Glutamax, 5 µg/ml of BDNF, 5 µg/ml of CNTF, and 0.5% Pen-Strep. A day after plating (day 2), neurons were supplemented with 10 ng/µl of AraC and incubated overnight.
nucleic acid extraction protocol
Total RNA was isolated from cells using RNeasy Mini Kit (Qiagen) followed by DnaseI (Qiagen) treatment.