Supplementary Figure 1: RNA-seq sample filters and quality control, including outlier detection, sex concordance, and assay performance measures. | Nature Neuroscience
a, Schematic of RNA-seq sample filtering. IDs of excluded samples are listed under each filtering step in gray text. Four QC tests were performed to identify outlier samples based on systematic abnormalities in overall expression (b,c,d). Moreover, we tested for sex concordance to identify potential sample mix-ups (e). b, Dendrogram visualizing pairwise Spearman correlations between gene expression levels of individual neuronal and non-neuronal samples. c, Histogram of median pairwise k-mer distances for each of the 115 samples with all other samples. d, Histogram of median pairwise Spearman correlations (D-statistics) between gene expression levels of individual samples. e, Concordance between clinical sex and sex-specific gene expression in neuronal and non-neuronal samples: normalized expression levels of the female-specific XIST transcript (x axis) and normalized expression levels of the Y-chromosome specific RPS4Y1 transcript (y axis) are shown. f, Scatterplot of two technical replicates based on lcRNAseq. N = 57,814; all annotated genes in GENCODE v19. g, Differential expression changes in linearly amplified RNA samples versus non-amplified RNA samples are preserved using qPCR consistent with previous reports validating the isothermal linear amplification method9,10. ACT values indicate the relative abundance of a target gene in one substantia nigra sample compared to one sample of human universal RNA. On the x-axis the relative abundance of the target gene in linearly amplified cDNA from one substantia nigra sample compared to linearly amplified cDNA from one sample of universal RNA is shown. One the y-axis, the relative abundance of the target gene in non-amplified cDNA from the same substantia nigra sample compared to non-amplified cDNA from the same sample of universal RNA is shown. The source RNA used for both the amplified and non-amplified experiment was identical. 25 target genes were analyzed by qPCR. h, lcRNAseq of melanized neurons from the SNpc highly enriches for dopaminergic markers genes consistent with previous reports (Poulin, J.F. et al. Cell Rep. 2014; Cahoy, J.D. et al. J. Neurosci. 2008). Relative expression abundance of dopaminergic genes (tyrosine hydroxylase, TH; dopamine transporter, SLC6A3; vesicular monoamine transporter, SLC18A1; dopamine receptor D2, DRD2) was highly enriched in laser-captured dopamine neuron samples compared to substantia nigra homogenates. By contrast, expression of microglia marker genes (purinergic receptor, P2RY12; protein tyrosine phosphatase receptor, PTPRC), astrocyte markers (glial fibrillary acidic protein, GFAP; connexin 30, GJB6), oligodendroglia markers (oligodendroglial transcription factors OLIG1 and OLIG2), and myelin markers (myelin oligodendrocyte glycoprotein, MOG; peripheral myelin protein, PMP22) was low in the laser-captured dopamine neuron samples compared to nigral homogenates.