Samples for microarray analysis were obtained in triplicate from separate coculture slips at 0, 0.5, 1, 6, 12, 36, and 48 h after ascorbic acid addition and were prepared for use on Illumina Mouse-8 chips (Illumina) by the Rockefeller Microarray Core Facility (New York, NY).
Growth protocol
Wild-type mice were obtained at embryonic day 13.5 for the extraction of DRG, which contain 2 main cell populations: Schwann cells and neurons. The DRG was disassociated and maintained until a dense layer of Schwann cells existed in tight proximity to neurons ( 9 days). The onset of Schwann cell–axonal maturation was triggered by adding ascorbic acid, defining time point zero
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from 10 to 20 mg of wet tissue using Qiagen RNeasy Mini kit according to the manufacturer's^ recommendations (Qiagen, Valencia, CA). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent Technologies Inc., Palo Alto, CA)
Label
Cy3
Label protocol
. ~500 ng of^ total RNA was used to prepare biotin-labeled RNA using Ambion Illumina TotalPrep RNA Amplification Kit (Cat# AMIL1791, Applied Biosystems, Foster City, CA).
Hybridization protocol
Briefly, 500 ng of^ total RNA was used to synthesize the first strand of cDNA using ArrayScript reverse transcriptase and an oligo(dT) primer bearing a T7 promoter. The single-stranded cDNA was then converted into a double-stranded DNA (dsDNA) by DNA polymerase^ I in the presence of /E. coli/ RNase H and DNA ligase. After column purification, the dsDNA was served as a template for in vitro transcription in a reaction containing biotin-labeled UTP, unlabeled NTPs and T7 RNA Polymerase. The amplified, biotin-labeled antisense RNA (aRNA) was purified and quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit. 750 ng of aRNA in 5 ul was mixed with 10 ul of hybridization reagents and heated at 65C for 10min. After cooled to room temperature, total 15 ul of the hyb solution was applied to Illumina MouseRef-8 v1.1 chip. The chip was incubated for about 18 hours at 58C. After washing and staining with streptavidin-Cy3, the chip was scanned using Illumina BeadArray Reader.
Scan protocol
The chip was incubated for about 18 hours at 58C. After washing and staining with streptavidin-Cy3, the chip was scanned using Illumina BeadArray Reader. The scanning was done using standard DirectHyb Gene Expression protocol with the following settings: Factor=1, PMT=587, Filter=100%.
Description
raw data samples 22-24
Data processing
The raw data was extracted using Illumina BeadStudio software without normalization.
