scRNA-seq of intestinal epithelial organoids derived from human fetal duodenum
scRNA-seq of intestinal epithelial organoids derived from human fetal duodenum
To dissociate organoids to single cells, matched healthy organoids for the same human sample in different media conditions were manually collected under a stereoscope. Following collection, dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, 130-092- 628) were used, and all incubation steps were carried out in a refrigerated centrifuge pre-chilled to 10°C unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in 1X HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 10°C with Mix 1 and then incubated for 10-minute increments at 10°C with Mix 2, and interrupted by agitation through pipetting with a P1000 pipette until fully dissociated. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 10°C and resuspended in 500mL 1X HBSS (with Mg2+, Ca2+). Cells were spun down (500g for 5 minutes at 10°C) and washed twice by suspension in 2 mL of HBSS + 1% BSA, followed by centrifugation. Cells were counted using a hemocytometer, then spun down and resuspended to reach a concentration of 1,000 cells/µL and kept on ice. Single cell libraries were immediately prepared on the 10x Chromium by the University of Michigan Advanced Genomics Core facility with a target capture of 5,000 cells. A full, detailed protocol of tissue dissociation for single cell RNA sequencing can be found at http://www.jasonspencelab.com/protocols. Duodenal tissue was cut into smaller pieces (~0.5-1 cm) and cut open longitudinally to expose the villi. To separate the epithelium, specimens then were incubated in disease(StemCell Technologies, Cat#07923) for 30 minutes on ice in a Petri dish. Dispase then was removed and replaced with 100% fetal bovine serum (FBS) for 15 minutes on ice. To mechanically separate the tissue layers, 3 mLs of Advanced DMEM/F12 (Gibco, Cat#12634-028) equal to the initial volume of FBS was added to the biopsy tissue before vigorously pipetting the mixture several times. Epithelial fragments then settled to the bottom of the Petri dish where they were collected manually on a stereoscope by pipette. The epithelium was then washed with ice-cold Advanced DMEM/F12 and allowed to settle to the bottom of a 1.5 mL tube. The supernatant was withdrawn from the loose tissue pellet and replaced with Matrigel (Corning, Cat# 354234). The Matrigel containing the isolated epithelium then was gently mixed before being pipetted into individual 50 µL droplets in a 24-well plate. The plate containing the droplets then was incubated at 37°C for at least 10 minutes to allow the Matrigel to solidify before adding media. Culture media consisted of LWRN conditioned media generated as previously described45,46 and combined with human basal media (Advanced DMEM/F12 (Gibco, Cat#12634-028); Glutamax 4mM (Gibco, Cat#35050-061); HEPES 20 mM (Gibco, Cat#15630-080); N2 Supplement (2X)(Gibco, Cat#17502-048), B27 Supplement (2X) (Fisher, Cat#17504-044), PenicillinStreptomycin (2X) (Gibco, Cat#15140-122), N-acetylcysteine (2 mM) (Sigma, Cat#A9165-25G), Nicotinamide (20 mM) (Sigma, Cat#N0636-061)). Complete media was comprised of 25% LWRN and 75% human basal media to which rhEGF (R&D, Cat#236-EG, 100 ng/mL) or rhEREG (R&D, Cat#5898-NR-050, 1 ng/mL) was added.  Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used