Drosophila Midgut scRNA-seq — Zipper et al. (eLife 2022)

R / Bioconductor Single-Cell Objects (.rds) and Expression Matrices

In the original analysis scripts, relabeling Cluster "2" to dEC (differentiating enterocytes) and merging the qISC (n = 104), pISC (n = 102), and EB (n = 274) progenitor annotations occurred at script runtime. Both pre-annotated objects and the original unmodified files are provided below.

File	Class & Dimensions	Description	Download
`Zipper2022_Midgut_Seurat_Annotated.rds`	`Seurat` 10,605 × 16,986 (RNA) / 10,141 (integrated)	Whole-midgut Seurat object (`RNA`, `SCT`, `integrated`; `pca`, `umap`) with `zipper2022_broad_celltype` (Cluster `"2"` → `"dEC"`) and `zipper2022_celltype` (`qISC`, `pISC`, `EB`, `dEC`, `aEC`, `mEC`, `pEC`, `EE`).	Download (152 MB)
`Zipper2022_ISC_EB_Imputed_Seurat_tSNE.rds`	`Seurat` 651 × 16,827	Imputed `ISC/EB` Seurat object with `tsne` reduction, `slingshot_Identity` (`qISC`, `pISC`, `EB`), and `marker_subtype`.	Download (23 MB)
`Zipper2022_Midgut_Imputed_SCE_Annotated.rds`	`SingleCellExperiment` 10,605 × 16,827	Imputed and deconvolution-normalized SCE object with `zipper2022_celltype` plus `all_genes_tSNE` and `metabolic_tSNE` coordinates.	Download (336 MB)
`trajectory-sce_ISC_EB.rds`	`SingleCellExperiment` 480 × 10,141	Dissected progenitor Slingshot trajectory object: `qISC` (104) → `pISC` (102) → `EB` (274).	Download (632 KB)
`slingshot-trajectory_ISC_EB.rds`	`SingleCellExperiment` 480 × 10,141	Slingshot curves and pseudotime object for `qISC → pISC → EB`.	Download (870 KB)
`trajectory-sce_diff.rds`	`SingleCellExperiment` 5,913 × 10,141	Whole-midgut 4-lineage differentiation trajectory object (`ISC/EB → dEC → pEC/mEC/aEC/EE`).	Download (67 MB)
`slingshot-trajectory_diff.rds`	`SingleCellExperiment` 5,913 × 10,141	Companion Slingshot pseudotime and 4 lineage curves for whole-midgut differentiation.	Download (74 MB)
`Deconvolution_tpm.rds`	`matrix` 16,827 × 10,605	`scran` deconvolution-normalized TPM matrix for KEGG metabolic pathway activity scoring.	Download (98 MB)
`selected_impute_ISC_EB_sce.rds`	`SingleCellExperiment` 651 × 16,827	Original imputed `ISC/EB` SCE object prior to subtype merge.	Download (24 MB)
`integrated.rds`	`Seurat` 10,605 × 16,986 (RNA) / 10,141 (integrated)	Original Seurat v3 integrated object prior to relabeling Cluster `"2"` → `"dEC"`.	Download (152 MB)
`selected_sce.rds`	`SingleCellExperiment` 10,605 × 16,827	Original whole-midgut SCE object prior to imputation.	Download (110 MB)
`tsne_final_ISC_EB.csv`	`CSV` 651 × 16,829	Imputed `ISC/EB` expression matrix and `tSNE_1, tSNE_2` coordinates.	Download (56 MB)
`Zipper2022_Scripts_Figures_Shiny_Package.zip`	`ZIP`	Complete archive of R scripts, `reproduce_figures.R`, Shiny `app.R`, metadata CSVs, `Methods.docx`, and SVG/JPEG figures.	Download Archive

Clustering Summary (vs. Hung et al. 2020, GSE120537)

Differentiating Enterocytes (dEC, Cluster "2"):
Cluster "2" (806 cells in integrated.rds; 653 cells in selected_impute_sce.rds) was unannotated in Hung et al. (2020). Metabolic gene clustering and Slingshot lineage reconstruction identified Cluster "2" as differentiating enterocytes (dEC) linking ISC/EB progenitors to aEC, mEC, and pEC.
Progenitor Dissection (qISC, pISC, EB):
Using scImpute dropout imputation and scran deconvolution normalization on the 651 ISC/EB cells, progenitors were resolved into:
- qISC (quiescent ISCs, n = 104): esg > 0, Dl < 1, klu < 1
- pISC (proliferating ISCs, n = 102): Dl ≥ 1, klu < 1
- EB (enteroblasts, n = 274): Dl < 1, klu ≥ 1

Usage in R

Run the Shiny app (all 16,827 genes):

shiny::runGitHub("Drosophila_midgut_metabolism", "Batchu-Sai")

Load annotated Seurat objects:

library(Seurat)

midgut <- readRDS("Zipper2022_Midgut_Seurat_Annotated.rds")
DimPlot(midgut, group.by = "zipper2022_celltype", label = TRUE)

isc_eb <- readRDS("Zipper2022_ISC_EB_Imputed_Seurat_tSNE.rds")
FeaturePlot(isc_eb, features = c("esg", "Dl", "klu", "whd"), reduction = "tsne")

ISC/EB Imputed tSNE (651 cells)

Select up to 6 genes to plot single-gene expression or pairwise co-expression (`Gradient_Plots/Plots.R`, `Plots_2025.R`).

Plot Mode: Add Gene:

Selected Genes:

Co-Expression Quantile Threshold: 0.30

Gene Panels:

Slingshot Trajectory: qISC → pISC → EB

480 progenitor cells ordered along Slingshot pseudotime (qISC n=104, pISC n=102, EB n=274).

Add Gene:

Whole-Midgut tSNE (5,631 Cells): All Genes vs. Metabolic Genes

Clusters: ISC/EB, dEC (Cluster 2), aEC, mEC, pEC, and EE (`TSNE_plots/Plots.R`).

Progenitor Subtype Mean Expression (`qISC`, `pISC`, `EB`, All `ISC/EB`)

Imputed log-normalized expression across qISC (n=104), pISC (n=102), EB (n=274), and all ISC/EB (n=651).

Download All 16,827 Genes (CSV) Download Key Panel (CSV)

Gene	Mean qISC (n=104)	Mean pISC (n=102)	Mean EB (n=274)	Mean All ISC/EB (n=651)	% Expr qISC	% Expr pISC	% Expr EB	Plot

Whole-Midgut Differentiation Trajectories (`ISC/EB → dEC → aEC/mEC/pEC/EE`)

Slingshot lineages bridging progenitors through Cluster 2 (dEC) and metabolic gene expression along pseudotime.

Dissected Progenitor Trajectory (`qISC → pISC → EB`)

Slingshot pseudotime trajectory of 480 progenitor cells and expression of fatty acid beta-oxidation / miR-277 target genes.

ISC/EB Progenitor Marker Blend (`Gradient_Plots/Plots.R`)

Blended expression of klu vs. Dl across 651 ISC/EB cells and esg vs. pros in qISC.

Single & Pairwise Co-Expression Grid (`Plots_2025.R`)

Co-expression grid on the 651-cell imputed ISC/EB tSNE embedding.

KEGG Metabolic Pathway Heatmap: `qISC` vs. `pISC` vs. `EB`

Metabolic pathway activity across quiescent ISCs, proliferating ISCs, and enteroblasts (`Dissection_Heatmaps.R`).

Combined Midgut Metabolic Pathway Heatmap (`qISC, pISC, EB, aEC, mEC, pEC`)

Metabolic pathway activity across progenitors and regional enterocytes.

Drosophila Adult Midgut scRNA-seq Clustering & Trajectory Data