Comprehensive Preprocessing of Sequencing Datasets

Full details of each dataset^{9 (link),10 (link),17 (link),20 ,21 (link),37 (link),42 ,50 (link),55 ,64 (link)–69 (link)}, including data type, sample type, source, and normalization approach, are available in Supplementary Table 1. Briefly, next generation sequencing datasets were downloaded and analyzed using the authors’ normalization settings unless otherwise specified; these consisted of transcripts per million (TPM), reads per kilobase of transcript per million (RPKM), or fragments per kilobase of transcript per million (FPKM) space. For analyses in log₂ space, we added 1 to expression values prior to log₂ adjustment. Affymetrix microarray datasets were summarized and normalized as described in ‘Gene expression profiling – Microarrays’ (Supplementary Note 1), using RMA in cases where bulk tissues and ground truth cell subsets were profiled on the same Affymetrix platform, and otherwise using MAS5 normalization. NanoString nCounter data were downloaded from the supplement of Chen et al.²⁰ and analyzed with batch correction in non-log linear space, but without any additional preprocessing.
Two publicly available PBMC datasets from healthy donors profiled by Chromium v2 (5’ and 3’ kits) were downloaded (Supplementary Table 1) and preprocessed as described in ‘Gene expression profiling – Single-cell RNA-seq’ (Supplementary Note 1), with the following minor modifications. During quality control, we excluded cells with >5000 expressed genes for 5’ PBMCs, >4000 expressed genes for 3’ PBMCs, and <200 expressed genes for both datasets. Seurat “FindClusters” was applied on the first 20 principal components, with the resolution parameter set to 0.6. Cell labels were assigned as described above. In addition, myeloid cells were defined by high CD68 expression, megakaryocytes by high PPBP expression, and dendritic cells by high FCER1A expression.
For the 3’ FL signature matrix in Supplementary Figs. 11d, and14a-b, publicly available 10x Chromium v2 scRNA-seq data (3’ kit)⁷⁰ were downloaded (Supplementary Table 1) and preprocessed as described for the 10x PBMC signature matrices above, but with the following differences. Seurat “FindClusters” was applied on the first 10 principal components, with the resolution parameter set to 0.6. Cell labels were assigned based on the following canonical marker genes (MS4A1 = B cells; CD3E, CD8A and CD8B = CD8 T cells; CD3E and CD4 = CD4 T cells).