Extended Data Figure 9: RNA-seq identification of candidate genes differentially expressed in NAc- and CeM-projecting BLA neurons.

a, Candidate differentially expressed genes were required to be enriched in only one group (either CeM or NAc projectors) in two independent experiments (NAc projectors collected from n = 8 mice; CeM projectors collected from n = 9 mice, total) at the indicated quantile fold-change threshold (light-blue column). One of the chance estimates (‘flip-flopped’, see Methods) is taken from genes that passed the quantile thresholds but were enriched in the opposite groups in the two experiments. Another chance estimate (‘permuted’, see Methods) is determined based on an analysis in which fold differences for each gene were permuted across genes within each of the two experiments before determining differential expression. A 0.02 quantile threshold was chosen to identify differentially expressed candidate genes in order to balance specificity and sensitivity, resulting in an estimated false discovery rate of 41.5%, calculated as the number expected by chance (flip-flopped) divided by the number of differentially expressed genes (see Extended Data Fig. 9c for candidate gene list). In Fig. 4k, a 0.01 quantile threshold was chosen to identify a more conservative list of differentially expressed candidate genes at a lower false discovery rate of 26.2%. b, Distribution of differentially expressed genes between NAc and CeM projectors from RNA-seq experiments 1 and 2 (see Methods). Light-blue shaded areas represent the 2nd and 98th percentiles of the distributions. c, RNA-seq heat map showing normalized expression levels of differentially expressed genes in NAc- and CeM-projecting BLA neurons. Differentially expressed genes were required to be enriched in either NAc or CeM projectors in two independent experiments (samples used in experiment 1 are indicated in black text below the heat map; experiment 2 samples are indicated in blue text) at a 0.02 quantile threshold (Extended Data Fig. 9a). Each RNA-seq library was prepared from 35–60 manually sorted retrobead-labelled cells taken from the BLA.