Figure 3. TNEs signatures accurately cluster dopamine and pyramidal neurons.

a, Of the 37,007 pyramidal neuron TNEs discovered, 34,077 (92.1%) were replicated in a second cohort of pyramidal neurons (PY) laser-captured from layer V/VI of 36 new human autopsy brains that had not been used for the discovery study. Moreover, using our TNE detection pipeline, we deconvoluted expression of 14,679 (39.7%) and 10,718 (29%) of pyramidal neuron TNEs in two ribodepleted total RNA-seq datasets representing cortex homogenates from 498 and 795 individuals, respectively. AMP-AD, Accelerating Medicines Partnership–Alzheimer’s Disease Consortium; MSBB, Mount Sinai VA Medical Center Brain Bank; psychENCODE BrainGVEX (https://www.synapse.org/#!Synapse:syn4590909); b, Cell type preferential expression of three dopamine neuron-exclusive TNEs, three pyramidal neuron-exclusive TNEs, and one TNE common to both dopamine and pyramidal neurons (in intron 4 of the PD gene SNCA; see also Supplementary Fig. 6a) was confirmed additionally by qPCR (box plots, top panel) in addition to lcRNAseq (pile graphs, bottom panels; and Supplementary Fig. 6b). Relative mRNA abundance of a classical dopamine-neuron marker, the dopamine transporter gene SLC6A3 (DAT), was assayed as positive control. Dopamine neuron samples (DA), N = 3; temporal cortex pyramidal neurons (TCPY), N = 3; primary human fibroblasts (FB), N = 2; human peripheral blood mononuclear white cells (PBMC), N =2. The pile graph tracks show RNA-seq read densities in dopamine neurons (DA), pyramidal neurons (PY), and non-neuronal cells (NN), as well as corresponding histone enhancer marks and transcription factor (TF) ChIP-seq peaks from ENCODE²⁵. Box plots indicate the median (bold line), the 25th and 75th percentiles (box edges), and the most extreme data point which is no more than 1.5 times the interquartile range from the box (whiskers). c, A signature based on cell type-exclusive TNE clustered 106 individual samples with 99.1% accuracy by t-SNE. d, The heatmap visualizes normalized counts for the 100 most abundant TNEs exclusively expressed in dopamine neuron (DA), pyramidal neuron (PY), and non-neuronal cells (NN).