RNAseq of small intestinal organoids prepared from wild type mice and mice harbouring a mutation in Gucy2c at position Serine 839 that converts it to an Isoleucine, followed by treatment with uroguanylin
Organoids were cultured for 3 days prior to treatment with uroguanylin (1 micro molar) or medium alone. 18h following treatment, organoids were harvested in DMEM:f12 medium and RNA prepared. Organoids were cultured for 3 days prior to RNA extraction Organoids prepared from wild type and S839I mice were cultured for 3 days after passaging and then treated with uroguanylin for 16h. Total RNA purified form cultured organoids using Invitrogen RNA purification kits. RNA was treated with DNAse on the column. mRNA fragment and synthesis of cDNA followed by end repair and dA tailing. cDNA purified and amplified by PCR