We converted original bcl format of sequences to fastq files using cellranger mkfastq. Alignment of the reads by cellranger count to the rheMac10 genome using the custom rheMac10 gtf yielded 61,609 and 23,690 nuclei for monkey P and F, respectively. We used a Seurat v3 pipeline to perform an integrated analysis of monkey F and P (85,299 nuclei in total). First, we removed ambient RNA in monkey P using SoupX.73 We then deleted ribosomal genes for both monkeys, and removed doublets using DoubletDetection,74 which lowered the number of nuclei to 80,902. Neuronal cells express higher numbers of genes compared to non-neuronal cells62,75,76 and therefore we used two different thresholds to remove low quality neuronal and non-neuronal nuclei. We chose these thresholds as the minimal level that produced clear cluster separation (Figure 1E). A total of 31,258 genes were identified, with an average of 3,200 per nucleus. We performed standard log-normalization and a variance stabilizing transformation prior to finding anchors and identified variable features individually for each monkeys’ data set using Seurat’s FindVariableFeatures function with number of features set to 7000.
