title: Illumina NovaSeq 6000 sequencing; scRNA-seq of pluripotent stem cell-derived human intestinal organoids grown in Epiregulin
design: scRNA-seq of pluripotent stem cell-derived human intestinal organoids grown in Epiregulin
library_name: 100ng/mlEGF_HIO_s
library_construction_protocol: To dissociate human intestinal organoids to single cells, organoids were mechanically isolated from Matrigel droplets and then tissue minced into small fragments using a scalpel in a petri dish filled with ice-cold 1X HBSS (with Mg2+, Ca2). This tissue was then transferred to a 15 mL conical tube. Dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, cat. no. 130-092-628) were used, and all incubation steps were carried out in a 37ºC incubator unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 37ºC with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 37ºC until digestion was complete. After each 10 minute incubation, tissue was agitated using a P1000, and tissue digestion was visually assessed under a stereo microscope. This process continued until the tissue was fully digested. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 4ºC and resuspended in 500µl 1X HBSS (with Mg2+, Ca2+). Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL and kept on ice. Single cell droplets were immediately prepared on the 10x Chromium according to manufacturer instructions at the University of Michigan The Advanced Genomics Core, with a target of capturing 5,000 cells. Single cell libraries were prepared using the Chromium Next GEM Single Cell 3' Library Construction Kit v3 according to manufacturer instructions. 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.  Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v3, 16 rxns PN-120237 was used Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v3, 16 rxns PN-120237 was used