Illumina HiSeq 2500 sequencing; Bulk RNA-seq of Adult and Fetal Enteroids (Control or Treated with DBZ)
Bulk RNA-seq of Adult and Fetal Enteroids (Control or Treated with DBZ)
Fresh human fetal epithelium was isolated and maintained as previously described (Tsai et al. 2018, Supplemental Protocol 1). Once enteroids were established, healthy cystic enteroids were manually selected under a stereoscope, bulk-passaged through a 30G needle, and embedded in Matrigel(Corning, 354234). Control culture media consisted of 50% LWRN conditioned media (Miyoshi & Stappenbeck, 2013) and 50% Human 2X 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)]. NOTCH inhibition media was control media with the addition of 5 μM DBZ Dibenzazepine; Apexbio Technology LLC; YO-01027). "Enteroids were grown in either control media as mentioned in \""growth protocol\"" and experimental group was treated with 5 μM DBZ in control media. " RNA from each sample was isolated using MagMAX-96 Total RNA (AM1830; Applied Biosystems), RNA isolation kits.  Input for library generation with Takara SMARTer Stranded Total RNA Sample Prep Kit (634876; Takara Bio USA)