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 and maintained as previously described (Tsai et al., 2018). 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). 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) was supplemented. Prior to scRNA-seq, EGF (100ng/ml) was either maintained (controls) or replaced with NRG1 (100 ng/ml) in the culture media for 5 days.  After 5 days, enteroids were collected and processed for scRNA-seq 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.