title: snRNA-seq of pluripotent stem cell-derived human intestinal organoids grown in vitro and in vivo with Epiregulin
design: snRNA-seq of pluripotent stem cell-derived human intestinal organoids grown in vitro and in vivo with Epiregulin
library_construction_protocol: Organoids were retrieved from their cultures or from their murine host and immediately snap-frozen for future nuclear extraction.  Maintenance and differentiation into HIOs were carried out as previously described. Cells were kept in a 37°C tissue culture incubator with 5% CO2 and lines were maintained in mTeSR Plus cultured media (Stemcell Technologies Cat# 100-1130). Stem cells underwent directed differentiation into definitive endoderm over a 3-day treatment using Activin A (100ng/mL, R&D Systems Cat#338-AC) added to RPMI base media. This base media was supplemented with 0%, 0.2%, 2% HyClone dFBS (Thermo Fischer Cat#SH3007103) on subsequent days with the addition of 5 mL penicillin-streptomycin each day (Gibco Cat# 15070063). After three days, endoderm monolayers were differentiated into an intestinal identity by treatment with FGF434 (500ng/mL) and CHIR99021 (2μM, APExBIO Cat#A8396). On days 4-6 of hindgut differentiation, spheroids budded from the monolayer and were collected. These spheroids were embedded in Matrigel as previously described32 and maintained in basal growth media consisting of Advanced DMEM/F12 (Gibco Cat# 11320033) with B27 (50x, Thermo Fisher Cat#17504044), GlutaMAX (1X, Gibco Cat#35050061), penicillin-streptomycin (Gibco Cat# 15070063), and HEPES buffer (15 mM , Gibco Cat#15630080). Organoid basal growth media was supplemented with epidermal growth factor (EGF) (100 ng/mL, 10 ng/mL, 1 ng/mL R&D Systems Cat#236-EG-01M) or Epiregulin (EREG) (100 ng/mL, 10 ng/mL, 1 ng/mL R&D Systems Cat#1195-EP-025/CF) with Noggin-Fc (100ng/mL, purified from conditioned media35), and R-Spondin1 (5% conditioned medium36) for the first three days of culture to pattern a proximal small intestine. On the third day after embedding, media was changed to basal growth media supplemented with EGF or EREG only (no additional Noggin or R-Spondin1) and remained in this media for the duration of the experiments with media changes every 5 days. Organoids were not passaged to avoid disrupting the development and spatial organization of the key cell types seen in EREG-grown HIOs. After at least 28 days, organoids were transferred into the kidney capsule of a mouse and allowed to grow for 10-12 weeks before harvesting.  Nuclei were isolated and permeabilized in accordance with 10x Genomics' Chromium Nuclei Isolation Kit Protocol (10x Genomics Cat#1000493). Briefly, tissue was minced into smaller fragments and then placed in lysis buffer where it was further dissociated mechanically with a pellet pestle. Tissue was then incubated in the lysis buffer for 5-7 minutes. The suspension was passed through the nuclei isolation column and spun at 16,000g for 20 seconds at 4°C. The suspension was then vortexed for 10 seconds and centrifuged at 500g for 3 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 500 µL of Debris Removal Solution and centrifuged at 700g for 10 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 1 mL of Wash Solution and centrifuged at 500g for 5 minutes at 4°C twice. The final pellet was resuspended in diluted nuclei buffer. Nuclei capture was carried out on the 10X Chromium platform with a target capture of 5000 nuclei per sample, and libraries were immediately prepared by the University of Michigan Advanced Genomics Core facility. Nuclei capture was carried out on the 10X Chromium platform with a target capture of 5000 nuclei per sample, and libraries were immediately prepared by the University of Michigan Advanced Genomics Core facility.