Dictionary of human intestinal organoid responses to secreted niche factors at single cell resolution

For scRNA-seq experiments, organoids were dissociated

Section Single cell preparation of organoids for single cell RNA-sequencing, offset —

For the perturbation dataset, the 10x 3’ high throughput kit was used.

Section Single cell preparation of organoids for single cell RNA-sequencing, offset —

loaded onto a 10X Chromium X according to manufacturer instructions

Section Single cell preparation of organoids for single cell RNA-sequencing, offset —

Sequenced reads were aligned using the human reference genome GRCh38-2020-A

Section scRNA-seq data pre-processing, offset —

via donor-pooled, multiplexed single cell RNA-sequencing (scRNA-seq)

Section series_summary, offset —

Sections of human transverse colon were provided by Donor Network West from deceased patients with no history of intestinal disease.

Section Generation of primary human colon organoids, offset —

from deceased patients with no history of intestinal disease

Section Generation of primary human colon organoids, offset —

Sections of human transverse colon were provided by Donor Network West from deceased patients with no history of intestinal disease.

Section Generation of primary human colon organoids, offset —

Organoids were collected for scRNA-seq on day 8 post-seeding.

Section Secreted niche factor perturbation screen, offset —

For the perturbation atlas, organoids from 2 female donors and 1 male donor were used.

Section Generation of primary human colon organoids, offset —

human recombinant proteins corresponding to the 79 selected perturbations (Supplementary Data 13 ) were applied to each well in an arrayed format.

Section Secreted niche factor perturbation screen, offset —

we generated a molecular dictionary of epithelial responses to secreted niche factors in human colon organoids across 3 donors via multiplexed scRNA-seq

Section Landscape of colonic epithelial cell responses to secreted niche factors at single cell resolution, offset —

index.html

Section filelist.txt, offset —

Supplementary files format and content: Processed Seurat objects (.Robj)

Section samples, offset —

scRNA-seq data was processed using CellRanger and resulting count matrices were analyzed using (v5.0.2) in Rstudio (v4.2.1).

Section scRNA-seq data pre-processing, offset —

Public on Dec 11 2025

Section series, offset —

the souporcell algorithm was used to distinguish between organoids from unique donors

Section scRNA-seq data pre-processing, offset —

Supplementary files format and content: Processed Seurat objects (.Robj)

Section samples, offset —

Supplementary files format and content: Processed Seurat objects (.Robj)

Section samples, offset —

GSE313368_RAW.tar

Section filelist.txt, offset —

Doublets were removed from data using the DoubletFinder algorithm

Section scRNA-seq data pre-processing, offset —

data was batch-corrected using the Harmony algorithm

Section scRNA-seq data pre-processing, offset —

Cell-type annotation was performed by applying FindTransferAnchors and TransferData using our matched colonic tissue reference atlas, and then manually cleaning up annotations

Section Cell type annotation in organoids, offset —

Doublets were removed from data using the DoubletFinder algorithm

Section scRNA-seq data pre-processing, offset —

resulting count matrices were analyzed using (v5.0.2) in Rstudio (v4.2.1)

Section scRNA-seq data pre-processing, offset —

After filtering and quality control, we obtained 169,996 cells

Section Landscape of colonic epithelial cell responses to secreted niche factors at single cell resolution, offset —

Sections of human transverse colon were provided by Donor Network West

Section Generation of primary human colon organoids, offset —

low-dose cytomix (10 ng/mL TNFɑ, 10 ng/mL IFNɣ, 1 ng/mL IL1β) was applied to the organoids

Section Secreted niche factor perturbation screen, offset —

After filtering and quality control, we obtained 169,996 cells

Section Landscape of colonic epithelial cell responses to secreted niche factors at single cell resolution, offset —

After filtering and quality control, we obtained 169,996 cells with 3 donors ×83 perturbations, for a total of 249 independent conditions.

Section Landscape of colonic epithelial cell responses to secreted niche factors at single cell resolution, offset —

the souporcell algorithm was used to distinguish between organoids from unique donors

Section scRNA-seq data pre-processing, offset —

We developed 4 epithelial injury models starting from homeostatic primary colon organoids derived from 3 human donors.

Section Development of a human colonic organoid injury model that recapitulates molecular features of human IBD, offset —