Fig. 2

MN CLIP generated a bona fide and robust MN NOVA binding profile. a Autoradiogram showing NuPAGE separation of radio-labeled GFP-NOVA2–RNA complexes. Both the transgene and UV crosslinking were required for the presence of radio-labeled GFP-NOVA2–RNA complex (lanes 1–3), which appeared as a smear from 100 to 125 kD with partial RNase digestion and collapsed to a band around 95 kD in high RNase concentration (lanes 3 and 4). b Representative RT-PCR polyacrylamide gel images for CLIP library cloning. Left panel shows RT-PCR products at incremental PCR cycle numbers and right panel shows control reactions without reverse transcriptase. The red box indicates the cDNA (with 5′ and 3′ linkers) size range purified for subsequent cloning and sequencing. c Pairwise correlation of normalized CLIP peak height (PH) between #6 and #17 transgenic lines and between two WSC groups. Normalized PH is the sum of raw CLIP reads in a given peak normalized to the read depth of each individual experiment. For comparison purpose, CLIP reads in WSC groups were randomly downsampled in the same number of peaks (27,628) to match the complexity of MN CLIP. d Enrichment of YCAY around CLIP peaks. YCAY enrichment is calculated by normalizing the number of YCAY motifs starting at a given position relative to all WSC or MN CLIP peaks to the expected YCAY frequency based on random base distribution. e Molecular function GO enrichment of MNs relative to WSC NOVA targets. FDR values were calculated using hypergeometic test, followed by Benjamini-Hochberg multiple test correction. The top six GO terms with the smallest FDR values are shown. Horizontal bars shows significance of GO enrichment, with significantly enriched GO terms in green, and nonenriched in gray. Dashed line marks the FDR value 0.1. f Volcano plot of gene-wise CLIP read enrichment in MNs versus WSC. Illustration of spinal cord transverse section is shown on the top right corner, with Rexed laminae I–VIII in purple and lamina IX in green. Genes with a restricted expression pattern in Rexed lamina IX and laminae I–VIII, as curated by the Allen Spinal Cord Atlas, are shown as green and purple dots, respectively. All other NOVA CLIP targets are displayed as gray circles. Two MN and two interneuron marker genes are labeled. The numbers of NOVA CLIP targets with enriched or depleted NOVA binding in MNs are indicated at the upper right and left corners of the chart, respectively, with font colors indicating the Allen Spinal Cord Atlas curated subgroups. The horizontal blue dashed line denotes FDR value 0.01