Illumina NovaSeq 6000 paired end sequencing; scRNA-seq of human fetal duodenal enteroids grown in EGF and/or NRG1
scRNA-seq of human fetal duodenal enteroids grown in EGF and/or NRG1
An established duodenal enteroid line derived from a 20.2 week post conception biological specimen was exposed to either EGF or NRG1 for five days Fresh human fetal epithelium was isolated as previously described (Tsai et al., 2018). Enteroid lines were derived in the presence of NRG1 (100ng/ml) or both NRG1 (100 ng/ml) and EGF (100 ng/ml) in addition to the standard EGF (100ng/ml) conditions in complete LWRN media. Once enteroids were established, healthy cystic enteroids were manually selected under a stereoscope and bulk-passaged through a 30G needle and embedded in Matrigel (Corning, 354234). Complete culture media consisted of LWRN conditioned media generated as previously described (Miyoshi and Stappenbeck, 2013; Tsai et al., 2018) and human basal media [Advanced DMEM/F12 (Gibco, 12634-028); Glutamax 4 mM (Gibco, 35050-061); HEPES 20 mM (Gibco, 15630-080); N2 Supplement (2X) (Gibco, 17502-048), B27 Supplement (2X) (17504-044), Penicillin-Streptomycin (2X) (Gibco, 15140-122), N-acetylcysteine (2 mM) (Sigma, A9165-25G), Nicotinamide (20 mM) (Sigma, N0636-061)]. Complete media was comprised of 25% LWRN and 75% human basal media to which rhEGF (R&D, 236-EG, 100ng/ml) and/or rhNRG1 (R&D, 5898-NR-050, 100ng/ml) was added. Enteroids were sequenced at passage 2.   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.