ER_nomusc_m1277
Protocols: Organoids (see growth protocol) wells were first disrupted by strong pipetting and pelleted before lysis. Colon tissue was used for muscle isolation. After flushing with cold PBS, colons were opened in longitudinal and dissected under a bench scope. Using a coverslip, mucosal tissue was completely removed carefully under the dissection scope and muscle was cut in 2-4 cm pieces, wash with sterile PBS twice, spin down and collected in P24 well plates. Pieces of muscle were inspected under the microscope for residual crypts or fat and further cleaned. Pieces were then incubated in DMEM-F12 for 24h before collecting the supernatant (muscle-SN). Muscle-SN was filtered (0,20um) and used directly + ENR factors in organoids cultures (right after splitting). Organoids were exposed to muscle-SN for 5 days. Muscle pieces were fixed in paraformaldehyde (PFA) 4% and processed in paraffin for H&E. Tissues were inspected to confirm the absence of mucosa. Small intestinal crypts were isolated and cultured following previously published protocol (Sato 2013). Organoids were cultured in basal crypt culture medium (BCM) consisting of advanced Dulbecco's modified Eagle medium -F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM Glutamax, 1x N2 [ThermoFisher Scientific 100X, 17502048], 1x B-27 [ThermoFisher Scientific 50X, 17504044], and 1x N-acetyl-L-cysteine [Sigma, A7250]) and overlaid with ENR factors containing 50 ng/ml of murine EGF [Thermo Fisher Scientific, PMG8041], 20% R-Spondin-CM (conditioned medium), kind gift from Calvin Kuo, Stanford University School of Medicine, Stanford, CA, USA), and 10% Noggin-CM. Medium was renewed every other day. For passaging, organoids cultures were washed, and matrigel and organoids were disrupted mechanically by strong pipetting, centrifuged at 200g, 5 min at 4 °C and resuspended in matrigel to plate in P24 wells. References Sato and Clevers. 2013. Primary Mouse Small Intestinal Epithelial Cell Cultures. Epithelial Cell Culture Protocols: Second Edition Organoids were cultured alone or exposed to muscle supernatant (see dissection protocol) for 5 days. Wells contained ENR control medium, ER (without Noggin factor), ENR + muscle supernatant (muscle-SN) or ER + muscle supernatant. RNA isolation was performed following manufacturer instructions (Direct-zol™ RNA MiniPrep, BioSite R-2052). RNA was quantified by spectrophotometry (ND1000 Spectrophotometer, NanoDrop, Thermo Scientific) and 25 ul at a 50 ng/ul concentration of RNA were used for RNA seq. Library preparation and sequencing for organoid RNA seq was performed by Novogene (UK) Co. NEB Next® Ultra™ RNA Library Prep Kit was used to generate the library.
ENA-FIRST-PUBLIC
2021-05-01T00:23:34Z
ENA-LAST-UPDATE
2020-06-03T20:02:38Z
External Id
SAMEA6872424
INSDC center name
Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
INSDC first public
2021-05-01T00:23:34Z
INSDC last update
2020-06-03T20:02:38Z
INSDC status
public
Submitter Id
E-MTAB-9181:ER_nomusc_m1277
age
12 to 15
broker name
ArrayExpress
cell type
epithelial cell of small intestine
common name
house mouse
developmental stage
adult
genotype
wild type genotype
growth condition
organoid culture without Noggin (ER medium)
individual
A
organism part
epithelium of small intestine
sample name
E-MTAB-9181:ER_nomusc_m1277
scientific_name
Mus musculus
sex
male
strain
C57BL/6