8 protocols
ID_REF = VALUE = Quantile normalized gene level expression values from RMA.
NFI-EnR data were processed using RMA. Gene expression values in the Affymetrix cel files were first background corrected and normalized together using RMA algorithm (Bioconductor R package). Low expression probes (i.e., expression value below 40th percentile in at least 7 out of 8 arrays) were removed. For multiple probes mapping to the same gene, only the probe showing the highest expression change for each gene was retained. Finally, moderated t-statistics and FDR correction (limma R package v.2.15.1.) were used to assess the significance of differential expression. Identical steps for signal normalization, background noise correction and statistical testing were applied to temporal arrays. NFI differentially regulated genes were defined as probes with FDR P-value<0.15 and fold change>1.5. Differentially expressed temporal genes were defined as having probes with FDR P-value<0.1 and fold change>2.
Affymetrix Gene ChIP Scanner 3000 7G
Samples were hybridized using the Affymetrix Hybridization Cocktail recipe consisting of components (BSA 50mg/ml from Invitrogen and Herring Sperm DNA from Promega) and lab prepared 2X Hybridization Buffer • Pre-hybridize arrays at 450C for 10 minutes with 1X Hybridization Buffer Heat cocktails at 99° for 5 minutes, then 45° for 5 minutes and centrifuge at max speed for ~3 minute (N.B. this deviates from Affy SOP)• Transfer 200 μl of hybridization solution to each array, then cover holes with Tough Spots • Hybridize ~16 hours at 45°C at 60rpm• Fluidics wash protocol used was EukGE-WS2v5.
Samples were prepared using an Affymetrix GeneChip 3’IVT Express Kit, in which total RNA undergoes reverse transcription to synthesize first strand cDNA and is then converted into double-stranded DNA template for transcription. IVT synthesizes aRNA and incorporates a biotin-conjugated nucleotide. The aRNA is then purified and fragmented.
RNA was extracted from cultured cells using Tri reagent (Sigma) and purified with an RNeasy Plus Micro Kit (QIAGEN).
Mouse CGN progenitors were purified from P6 mice cerebellum and cultured in complete Neurobasal medium until 1.5 or 6 days.
For NFI group, CGNs were transduced on the day of plating with lentiviruses expressing either NFI-EnR or EnR alone. Fresh medium was added on 3DIV (days in vitro).