Mus musculus
Protocols: 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.
strain
C57Bl6
replicate
3
disease
healthy
organism
Mus musculus
organism
Mus musculus
collection date
not collected
sex
male
scientific_name
Mus musculus
cell type
epithelial cell
common name
house mouse
organism part
duodenum
developmental stage
adult
genotype
wild type genotype
isolate
not applicable
cell line
organoid
age
166
geographic location (country and/or sea)
not collected