mutant 1.1
Mus musculus
Protocols: RNA was harvested by Trizol, DNA contaminations were removed by DNase1 digestion in the presence of Rnase Inhibitor, followed by RNA purification via phenol/chloroform extraction. For RNA Isolation, organoids were transferred to 15ml collection tube and washed once with ice cold PBS. RNA was extracted with Trizol, followed by DNA digestion. We established organoids from the small intestine of NICDEYFPflox/flox/p53flox/flox; VillinCreERT2 mice bred under sterile conditions. Small intestines were dissected and small intestinal crypt were isolated and cultured as 3D-intestinal organoids (as described in Sato et al. Nature 459, 262-265 (2009)). Mutagenesis were induced by 4-OHT-Tamoxifen (400nM for 24h) to generate mutant organoids. Organoids that served as wt controls were not treated with 4-OHT. Mutant organoids were selected by withdrawal of EGF, R-Spondin1 and Noggin. Mutant organoids were cultured in full medium prior comparing with wt-control organoids. Organoids were generated in independent experiments. RNA was harvested by Trizol, DNA contaminations were removed by DNase1 digestion in the presence of Rnase Inhibitor, followed by RNA purification via phenol/chloroform extraction. Sequencing libraries were generated using the Illumina TruSeq Stranded mRNA Kit and sample were processed in two pools with HiSeq 4000 in one run (1 pool - 1 lane).