Scalable Single-Cell RNA-seq Analysis

De-multiplexing, alignment to the mm10 transcriptome and unique molecular identifier (UMI)-collapsing were performed using the Cellranger toolkit (version 2.1.1, chemistry V2, or version 3.0.2, chemistry V3) provided by 10X Genomics for chemistry Single Cell 3’, and run using cloud computing on the Terra platform (https://Terra.bio). Since nuclear RNA is expected to include roughly equal proportions of intronic and exonic reads, we built and aligned reads to genome references with pre-mRNA annotations, which account for both exons and introns. For every nucleus, we quantified the number of genes for which at least one read was mapped, and then excluded all nuclei with fewer than 400 detected genes. Genes that were detected in fewer than 10 nuclei were excluded. Expression values E_i,j for gene i in cell j were calculated by dividing UMI counts for gene i by the sum of the UMI counts in nucleus j, to normalize for differences in coverage, and then multiplying by 10,000 to create TPM-like values, and finally computing log₂(TP10K + 1) (using the NormalizeData function from the Seurat^{22 (link)} package version 2.3.4).