Additional file 3 of McEnhancer: predicting gene expression via semi-supervised assignment of enhancers to target genes

McEnhancer predicted DHSs for each cluster. This table shows the predicted DHSs for each cluster, along with its associated gene. Columns represent DHS coordinates (chr, start, end), DHS ID linked to its associated gene, and number of times each DHS was selected in the model’s pairwise comparisons against other clusters. (TXT 778 kb

Additional file 1 of McEnhancer: predicting gene expression via semi-supervised assignment of enhancers to target genes

Supplementary figures. (PDF 9185 kb

Additional file 8: Table S2. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Top 50 marker genes expressed in 4366 sorted, fixed cells from mouse hindbrain and cerebellum. For explanations, see legend to Table S1. Related to Fig. 4. (XLSX 196 kb

Additional file 5: Table S1. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Top 50 marker genes expressed in 4873 fixed, primary cells from Drosophila embryos. Related to Fig. 3. Tables S1 and S2 contain the top 50 marker genes per cluster, provided by Seurat's function 'FindAllMarkers' [17]. We additionally ordered them per cluster in decreasing log2-fold change (log2FC). The log2FC was computed for a given gene by dividing its average normalized expression for a given cluster over the average normalized expression in the rest of the clusters and taking the logarithm of the fold change. (XLSX 214 kb

Additional file 2: Figure S2. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Fixed cell samples can be stored for weeks to give reproducible results. Related to Fig. 2. (a), (b) Drop-seq of mixed human and mouse cells (50 cells/μl), corresponding to a biological replicate of the experiment shown in Fig. 2. Libraries were sequenced to a median depth of ~142,400 (Fixed 1 week) or ~28,500 (Fixed 3 weeks) aligned reads per cell. (a) Plots show the number of human and mouse transcripts (UMIs) associated with a cell (dot) identified as human- or mouse-specific (blue or red, respectively). Cells expressing fewer than 3500 UMIs are grey. Both Drop-seq experiments yielded single-cell transcriptomes that allowed clear species separation and a low percentage of cell doublets. (b) Distribution and the median of the number of genes and transcripts (UMIs) detected per cell expressing more than 3500 UMIs. (c) Gene expression levels from live and fixed cells correlate well. Pairwise correlations between bulk mRNA-seq libraries and Drop-seq single-cell experiments for cells expressing more than 3500 UMIs. Non-single cell bulk mRNA-seq data are shown as reads per kilobase per million (RPKM). Drop-seq expression counts were converted to average transcripts per million (ATPM) and plotted as log2 (ATPM + 1). Upper right panel depicts Pearson correlations. The intersection (common set) of genes between all samples was high (~17,000 genes). (PDF 228 kb

Additional file 7: Figure S6. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Variance in single-cell data from newborn mouse hindbrain and cerebellum and 2D cluster representation of replicates. Related to Fig. 4. (a) Plots of principal components 1–18 of the 4366 cell transcriptomes show variance in many principal components. Colors correspond to tSNE plot in Fig. 4b. (b) 2D representation of experimental replicates in each cell population. tSNE plot from Fig. 4b with each cell now coloured by experimental replicate. Note that cells from the two biological replicates are unevenly represented in the different clusters, likely reflecting dissection differences and varying proportions of hindbrain to cerebellar tissue. (c) We identified a subtype of myelinating glia, probably Schwann cells from cranial nerves entering the hindbrain (cluster 11, Fig. 4b). These cells express myelin protein zero (Mpz) and other genes for myelin formation (proteolipid protein 1, Plp1) and Mbp (Fig. 4b) but do not express oligodendrocyte markers such as Bcas1 or Olig1 (Fig. 4b). (PDF 255 kb

Additional file 6: Figure S5. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Single-cell data from mouse hindbrain are reproducible and correlate well with bulk mRNA-seq data. Related to Fig. 4. (a) Identification of cell barcodes associated with single-cell transcriptomes for single-cell libraries from FACS-sorted, fixed mouse hindbrain cells. (For methods details, see Additional file 1: Figure S1). (b) Correlations between gene expression measurements from independent Drop-seq experiments with FACS-sorted methanol-fixed single cells (expressing >300 UMIs). Cells were from independent biological samples, representing dissected, dissociated mouse hindbrains and cerebellum from newborn mice. Bulk mRNA-seq data were generated with total RNA extracted from cells after FACS and fixation. Non-single cell bulk mRNA-seq data were expressed as reads per kilobase per million (RPKM). Drop-seq expression counts were converted to average transcripts per million (ATPM) and plotted as log2 (ATPM + 1). Upper right panel depicts Pearson correlations. The intersection (common set) of genes between samples was ~17,000 genes. (PDF 68 kb

Additional file 4: Figure S4. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Variance in single-cell data from Drosophila embryos and 2D cluster representations of replicates. Related to Fig. 3. (a) Plots of principal components 1–30 of the 4873 cell transcriptomes show variance captured in many principal components. Colors correspond to tSNE plot in Fig. 3b. (b) 2D representation of experimental replicates in each cell population. tSNE plot from Fig. 3b with cells now coloured by experimental Drop-seq replicate (left) or biological replicate sample (right). Clusters are formed by cells from many Drop-seq different runs (left) and from both samples (right). The relatively more homogenous composition of cluster 8 (neurons) and 15 (LVM) is consistent with a higher proportion of embryos of later stages in sample 2. (PDF 376 kb

Additional file 3: Figure S3. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Single-cell data from Drosophila embryos are reproducible and correlate well with bulk mRNA-seq data. Related to Fig. 3. (a) Identification of cell barcodes associated with single-cell transcriptomes for single-cell libraries from Drosophila embryos, a complex primary tissue harbouring small, low RNA content cells. (For methods details, see Additional file 1: Figure S1a.) Four of seven replicates are shown. (b) Correlations between gene expression measurements from bulk mRNA-seq and seven Drop-seq runs with methanol-fixed single cells (expressing >1000 UMIs). Cells were from two independent biological samples representing dissociated Drosophila embryos (75% stages 10 and 11). Bulk mRNA-seq data were generated with total RNA extracted directly from whole, intact, live embryos. (Sample 1: rep 1, 2, 7 and bulk 1; sample 2: rep 3–6 and bulk 2). Non-single cell bulk mRNA-seq data were expressed as reads per kilobase per million (RPKM). Drop-seq expression counts were converted to average transcripts per million (ATPM) and plotted as log2 (ATPM + 1). Upper right panel depicts Pearson correlations. The intersection (common set) of genes between all samples was high (~10,000 genes). (PDF 162 kb