Illumina NovaSeq 6000 sequencing; scRNA-seq of Human Alveolar Type 2 Cell Organoids
scRNA-seq of Human Alveolar Type 2 Cell Organoids
Organoids were dislodged from Matrigel by mechanical dissociation using a p1000 pipette tip. Pelleted organoids were resuspended in TrypLE Express (Thermo Fisher, Cat #17105041) and incubated at 37ºC, pipetting gently at 5 minute intervals until a single cell suspension is obtained (~10 minutes). Cells were washed 3x with Hanks Balanced Salt Solution (HBSS) (Thermo Fisher, Cat #14175095) + 1% BSA and then passed through a 40 µm FlowMi Cell Strainer. Cells were counted on a hemocytometer and then resuspended at 1000 cells/µl in HBSS + 1% BSA for submission to the the University of Michigan Sequencing Core, which prepared libraries using the Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1 (10x Genomics, Cat#PN1000128) targeting 3500 cells.  All organoids in this study were cultured in 8 mg/mL Matrigel growth matrix (Corning Cat#354234), fed every three days with indicated media and passaged into fresh Matrigel every 7 - 10 days by needle sheering.  Bud tip Progenitor Media (3F): DMEM/F-12 (Corning, Cat#10-092-CV), 100U/mL penicillin-streptomycin (Thermo Fisher, Cat#15140122), 1X B-27 supplement (Thermo Fisher, Cat#17504044), 1X N2 supplement (Thermo Fisher, Cat#17502048), 0.05% BSA (Sigma, Cat#A9647), 50µg/mL L-ascorbic acid (Sigma,Cat#A4544), 0.4 µM 1-Thioglycerol (Sigma, Cat#M1753), 50nM all-trans retinoic acid (Sigma, Cat#R2625), 10ng/mL recombinant human FGF7 (R&D Systems, Cat#251-KG), and 3µM CHIR99021 (APExBIO, Cat#A3011). CK + AB Media: DMEM/F-12 (Corning, Cat#10-092-CV), 100U/mL penicillin-streptomycin (Thermo Fisher, Cat#15140122),1X B27 without vitamin A (Thermo Fisher, Cat# 12587010) , 1X N2 supplement (Thermo Fisher, Cat#17502048), 0.05% BSA (Sigma, Cat#A9647), 50µg/mL L-ascorbic acid (Sigma,Cat#A4544), 0.4 µM 1-Thioglycerol (Sigma, Cat#M1753),  10ng/mL recombinant human FGF7 (R&D Systems, Cat#251-KG), and 3µM CHIR99021 (APExBIO, Cat#A3011), 1 µM A-8301 (APExBIO Cat#A3133), 100 ng/mL BMP4 (R&D Systems Cat#314-BP-050). CK + DCI Media: DMEM/F-12 (Corning, Cat#10-092-CV), 100U/mL penicillin-streptomycin (Thermo Fisher, Cat#15140122),1X B27 without vitamin A (Thermo Fisher, Cat# 12587010) , 1X N2 supplement (Thermo Fisher, Cat#17502048), 0.05% BSA (Sigma, Cat#A9647), 50µg/mL L-ascorbic acid (Sigma,Cat#A4544), 0.4 µM 1-Thioglycerol (Sigma, Cat#M1753),  10ng/mL recombinant human FGF7 (R&D Systems, Cat#251-KG), and 3µM CHIR99021 (APExBIO, Cat#A3011),50 nM Dexamethasone (Sigma, Cat#D4902), 100 nM 3-isobutyl-1-methylxanthine (Sigma, Cat#I5879) and 100 nM 8-Bromoadensoine 3',5'-cyclic monophosphate sodium salt (Sigma, Cat#B7880). SFFF without FGF10 Media: Advanced DMEM/F12, 2 mM Glutamax, 1X B27 supplement, 100 U/mL penicillin-streptomycin, 15 mM HEPES, 0.05% BSA, 10 µM TGF-inhibitor SB43152 (APExBIO, Cat#A8249), 1 µM p38 MAP kinase-inhibitor BIRB796 (APExBIO, Cat#A5639), 3 µM CHIR99021, 50 ng/mL rhEGF (R&D Systems, Cat#236-EG). Lung progenitor (bud tip) organoids were derived and maintained in vitro for at least 20 days prior to differentiation. For CK + AB and CK + DCI treatments, organoids were passaged to fresh matrigel, given three days to recover and then switched from Bud tip progenitor media to CK + AB or CK + DCI for 21 days, with passages occurring at days 7 and 14. At day 21, CK + AB or CK + DCI treated organoids were passaged to new matrigel and switched to SFFF without FGF10 for an additional 120 days. Immediately after organoid dissociation, single cell libraries were prepared on the 10x Chromium with a target of 3500 cells. Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics, Cat#PN1000128) following the manufacturer's protocol. Immediately after organoid dissociation, single cell libraries were prepared on the 10x Chromium with a target of 3500 cells. Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics, Cat#PN1000128) following the manufacturer's protocol.