RNA-Seq on mouse murine duodenal intestinal organoids/tumoroids exposed to conditioned media from duodenal intestinal smooth muscle explants
Organoids and tumoroids were seeded and continuously cultured until the experiment. Two days following seeding, the normal growth medium was replaced with growth medium that has been conditioned with muscle supernatant for 12-24 hours. Immediately before treatment and at 20 minutes, 2 hours, and 24 hours following treatment, wells were harvested by first replacing the media with ice-cold PBS, then scraping off the matrigel domes with a P1000 pipette tip. The organoid/tumoroid and matrigel slurry was dissociated by forcing the mixture against the wall of a 50mL conical vial and pelleted by centrifuging at 300 x G, 5 min, at 4C. The supernatant containing PBS and matrigel was then removed to obtain only the organoid/tumoroid pellet. For each RNA extraction, 3-4 wells of moderately seeded organoids or tumoroids (~100-200 per well) were pooled for one extraction. RNA was extracted using the QuickRNA Microprep kit from Zymo Research. Immediately after pelleting the organoid/tumoroids, 100uL of RNA lysis buffer from the QuickRNA Microprep kit was added to each pellet. The rest of the extraction was run in accordance with the instructions provided with the Zymo Research QuickRNA Microprep kit. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dTTP for a non-directional library. It was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.