Ncounter prep station

Extracted RNA was used as input for expression profiling by NanoString. All samples were prepared according to the manufacturer’s specifications (NanoString Technologies). Briefly, input RNA (100 ng) was hybridized with reporter probes to specific transcript tags in a multiplexed ligation reaction using reverse complementary bridge oligonucleotides. Spiked-in controls (in addition to housekeeping genes) were used in each reaction to permit normalization. After the ligation reaction, tagged transcripts were hybridized to tag-specific capture probes with attached fluorescent bar codes by incubation at 65°C for 14 to 28 hours. Excess probes were removed on the nCounter Prep Station according to the manufacturer’s instructions (NanoString Technologies), and the captured, transcript-specific, bar-coded ternary complexes were immobilized on a streptavidin-coated cartridge. The individual bar codes were counted on the nCounter Digital Analyzer (NanoString Technologies) using a high density of fields of view (>600) to quantify target RNA molecules present in the sample. Data are accessible in the GEO database under accession number GSE73094.

MiRNA expression was examined using total RNA isolated from 2 male rat cortices and 2 female rat cortices from each developmental stage. The RNA was analyzed using NanoString nCounter® Rat miRNA Expression Arrays (catalog #GXA-RMIR-12; NanoString Technologies, Seattle, WA). The NanoString miRNA panel contains 423 probes for rat miRNAs from miRBase 20. Total RNA (100 ng) was used as input for nCounter miRNA sample preparation reactions and the reactions were performed, as per the manufacturer’s instructions (NanoString Technologies). Small RNA sample preparation involves the ligation of a specific DNA tag onto the 3′ end of each mature miRNA. These tags normalize the melting temperatures of the miRNAs and provide a unique identification for each miRNA species in the sample. Excess tags were then removed, and the resulting material was hybridized with a panel of miRNA: tag-specific nCounter capture and barcoded reporter probes. Hybridized probes were then purified and immobilized on a streptavidin-coated cartridge using the nCounter Prep Station (NanoString Technologies). Data collection was carried out on the nCounter Digital Analyzer (NanoString Technologies) following manufacturer’s instructions to count individual fluorescent barcodes and quantify target RNA molecules present in each sample. For each assay, a high-density scan (600 fields of view) was performed.