All mice were healthy and were housed with a companion(s) in a specific-pathogen free animal facility in vented cages on a 12 hour light/dark cycle. Food and water were provided ad libitum.
Extracted molecule
total RNA
Extraction protocol
TRAP-RNA was isolated as previously described (Heiman et al., 2014; Kocabas et al., 2015; Zhu et al., 2016). Briefly, for Tg(Pcp2-L10a-Egfp) mice, pooled cerebella were immediately homogenized with a Teflon-glass homogenizer in ice-cold polysome extraction buffer (10 mM HEPES-KOH [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT (MilliporeSigma, D9779-1G), 100 µg/ml cycloheximide (MilliporeSigma, C7698-1G), Superasin and RNasin RNase inhibitors (ThermoFisher, AM2694, PR-N2515)and complete-EDTAfree protease inhibitor (MilliporeSigma, 11836170001)). Following clearing by centrifugation, supernatants were incubated at 4˚C with end-over-end rotation for 16-18 hours with biotinylated Streptavidin T1 Dynabeads (ThermoFisher, 65601) previously conjugated with GFP antibodies (Sloan Kettering Institute Antibody Core, HtzGFP-19C8 and HtzGFP-19F7). The beads were collected on a magnetic rack, washed, and resuspended in lysis buffer with β-mercaptoethanol (Agilent, 400753) to extract bound RNA from polysomes. RNA was purified using the Stratagene Absolutely RNA Nanoprep kit (Agilent, 400753). RNA quantity and quality was measured using an Agilent 2100 Bioanalyzer with the 6000 Pico Kit (Agilent, 5067-1513).
Label
biotin
Label protocol
300 ng of total RNA was used to synthesize the first strand of cDNA using GeneChip WT Plus Reagent Kit from Affymetrix(P/N 902281). Single-stranded cDNA with T7 promoter sequence at the 5’ end was synthesized in the reverse transcription reaction. 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. The dsDNA was then served as a template for amplification with T7 RNA polymerase to create antisense RNA. 15 μg of antisense RNA was used to generate sense-strand cDNA(ss-DNA) by reverse transcription using 2nd-Cycle Primers. The ss-cDNA contains dUTP at a fixed ratio relative to dTTP and 5.5 μg of ss-cDNA was fragmented by uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) at the unnatural dUTP residues. The fragmented ss-cDNA was labeled with biotin by terminal deoxynucleotidyl transferase (TdT).
Hybridization protocol
200 μl of hybridization solution containing 5.2 μg of the fragmented and labeled ss-DNA was hybridized to Affymetrix HTA 2.0 arrays (P/N 902662), (or GeneChip™ Mouse Gene 1.0 ST Array P/N 902464; GeneChip™ Mouse Gene 2.0 ST Array P/N 902462) for 16 hours at 45°C. After hybridization, arrays were stained with streptavidin-phycoerythrin, followed by an antibody solution (anti-streptavidin) and a second streptavidin-phycoerythrin solution,( used The GeneChip™ Hybridization, Wash, and Stain Kit P/N 900720) with all liquid handling performed by a GeneChip Fluidics Station 450. Arrays were then scanned with the Affymetrix GeneChip Scanner 3000 7G.
Scan protocol
Mouse Gene 1.0 ST arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Data processing
Normalised microarray expression values were acquired with Affymetrix Power Tools’s apt-probeset-summarize tool using rma-sketch normalisation, MoGene-1_0-st-v1.r3.pgf MoGene-1_0-st-v1.r3.mps