NextSeq 550 paired end sequencing; Single nucleus RNA sequencing of human iPSC-derived forebrain organoids to assay cell type composition
Single nucleus RNA sequencing of human iPSC-derived forebrain organoids to assay cell type composition
D1 spheroids were formed and cultured for 21 weeks under the conditions specified in the growth protocol. Subsequently, ca. 30 spheroids were frozen and single nuclei were extracted according to the protocol specified in the associated publication. Human iPSC lines D1 were cultured in mTeSR Plus medium (STEMCELL Technologies, Vancouver, Canada) according to the protocol provided by the manufacturer on Matrigel (Corning, Corning, NY, USA) coated cell culture plates. For forebrain organoid generation, the culture medium was supplemented with 10 µM ROCK inhibitor (Y-27632 dihydrochloride, Tocris, NY, USA) for 15 min followed by dissociation with Accutase (STEMCELL Technologies). 105 cells were transferred into each well of a low attachment conical bottom 96-well plate (ThermoFisher Scientific, Waltham MA, USA) and aggregated by centrifugation for 5 min at 75 x g. The next day, hiPSC aggregates were collected, taken up in neural induction medium (NIM: DMEM/F-12, 1 x GlutaMAX, 2 x B27 without Vitamin A, 1 x N2, β-Mercaptoethanol (#21985023, 1:1000) (ThermoFisher Scientific), Dorsomorphin dihydrochloride (500 nM, Tocris,), recombinant Noggin (5 ng/ml, R&D Systems, Minneapolis, MN, USA), SB431542 (10 µM, Tocris,), Normocin (1:1000, Invivogen, San Diego, CA, USA)) and transferred into non-coated 35 mm plastic dishes (#627102, Greiner, Kremsmünster, Austria). On day two in NIM, FGF-2 (10 ng/ml, Peprotech, Rocky Hill, NJ, USA) was added to the culture medium. After seven days in NIM, flee-floating organoids were moved into neural medium (NM: Neurobasal (ThermoFisher Scientific), 1 x GlutaMAX, 1 x B27 without vitamin A, Normocin) supplemented with FGF-2 (10 ng/ml) and EGF (10 ng/ml, Peprotech) for two weeks. In order to ensure forebrain fate adoption, IWP-2 (2 µM, Sigma Aldrich, St. Louis, MO, USA) and Cyclopamine (1 µM, Merck, Darmstadt, Germany) were included in the media for the first 21 days. From day 21 to 35 NM was supplemented with BDNF (10 ng/ml) and NT-3 (10 ng/ml, both Peprotech). From day 35 onwards organoids were kept in maintenance medium (MM: BrainPhys (STEMCELL Technologies), 1 x GlutaMAX, 1 x B27 without Vitamin A, Normocin).  Nucleic acids were processed using the 10x Chromium Single Cell 3' v3.1 Gene Expression Kit according to the manufacturer's instructions Libraries were constructed using the 10x Chromium Single Cell 3' v3.1 Gene Expression Kit according to the manufacturer's instructions
Experimental Factor: cell type
forebrain organoid cell