Differentiated gallbladder organoids were obtained by culturing the organoids in media lacking R-spondin 1, noggin and nicotinamide for two weeks.
Growth protocol
Organoids were cultured in Matrigel with tissue culture media based on AdDMEM/F12 (Life Technologies) and supplemented with B27, N2 (Life Technologies) and 1.25 uM N-Acetylcysteine (Sigma-Aldrich). The following growth factors were also added: 50 ng/mL murine recombinant EGF (Life Technologies), 50 ng/mL murine recombinant FGF10 (R&D system), 10 mM nicotinamide (Sigma-Aldrich), 50 ng/mL murine recombinant HGF (Peprotech) and R-spondin 1 and noggin home-made conditioned media (prepared as in Sato et al., Nature 459: 262-265, 2009).
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from liver, gallbladder or small intestine tissues, and liver, gallbladder or small intestine organoids using the RNeasy Mini RNA Extraction Kit (Qiagen).
Label
biotin
Label protocol
5.5 ug of ds cDNA were fragmented and labeled using the GeneChip WT DNA terminal labeling kit.
Hybridization protocol
Biotinylated cRNA was fragmented and hybridized to GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix) following the standard protocol of the manufacturer.
Scan protocol
Standard Affymetrix.
Description
LO2
Data processing
RMA Probes to Import: Interrogating Probes, Probe filtering: skip,Algorithm: RMA,Background Correction: RMA Background Correction,Normalization: Quantile Normalization,Log Probes using Base: 2,Probeset Summarization: Median Polish
