tissue: hindbrain, posterior to the pontine/hypothalamic junction; cerebellum removed rna type: TRAP Sex: both age: adult (6-12 weeks) number of mice: 5 line: B6;FVB-Tg(Slc6a2-EGFP/Rpl10a)JD1538Jdd/J
Extracted molecule
total RNA
Extraction protocol
Replicate pools of 5 mixed sex adult mice from each of the Slc6a2 lines were sacrificed and brains were removed and transferred to ice cold dissection buffer containing cycloheximide, and hindbrain, posterior to the pontine/hypothalamic junction was collected, discarding the cerebellum. TRAP was conducted as described (see PMID: 24810037). Briefly, each pool was homogenized for 12 strokes in a glass teflon homogenizer on ice, in buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 100 μg/ml cycloheximide, protease inhibitors, and recombinant RNase inhibitors). Nuclei and debris were removed with centrifugation at 2000g for 10 min at 4C. DHPC (Avanti, Alabastar, AL) and NP-40 (Ipgal-ca630, Sigma, St Louis, MO) were added to supernatant to final concentrations of 30mM and 1%, respectively. After 5 min incubation of ice, supernatant was centrifuged for 15 minutes at 20,000g, and pellet was discarded. Supernatant was mixed with protein G coated magnetic beads (Invitrogen/Life Technologies, Grand Island, NY), previously conjugated with a mix of two monoclonal anti-GFP antibodies,52 and incubated with rotation for 30 minutes at 4C. Beads were washed three times with high salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol, and 100 μg/ml cycloheximide) and RNA was purified from ribosomes using Trizol (Invitrogen), followed by DNAse treatment and further purification and concentration with Rneasy min-elute columns, all following manufacture’s protocol (Qiagen, Hilden, Germany). RNA was also harvested in parallel from each unbound fraction of affinity purification as a measure of total hindbrain RNA. RNA concentration of all samples was measured with Nanodrop spectrophotometer and integrity was confirmed with PicoChips on the Agilent BioAnalyzer (RIN>8).
Label
Biotin
Label protocol
Affymetrix two-cycle amplification kit
Hybridization protocol
Standard Affymetrix protocol for mouse 430 2.0 platform
Scan protocol
Standard Affymetrix protocol for mouse 430 2.0 platform
Description
RNA from immunoprecipitated ribsomes
Data processing
Data were analyzed using the Bioconductor module within the statistical package R. GCRMA was used to normalize within replicates, and to biotinylated spike in probes between conditions. Fold change, Specificity Index (SI) and pSI (see PMID: 20308160) were calculated for all genes with expression above non-specific background, and >50 arbitrary fluorescent units, as described. The threshold to identify genes expressed in the neurons above non-specific background was conservatively set at the mean plus 2 standard deviations of the fold change of negative control transcripts comparing the TRAP samples to hindbrain mRNA. Genes with expression below this level may or may not be found in LC neurons. To identify transcripts enriched in LC neurons compared to hindbrain RNA, we utilized the empirical Bayesian statistic with FDR correction within the Limma package. Hierarchical clustering was conducted in R utilizing the expression values from any genes with pSI <.01 in any cell type.
