IL13IFNG_M1_S5
Protocols: RNA was extracted (see nucleic acid extraction protocol) from small intestinal organoids (see growth protocol) treated with cytokines for 24 hours (see treatment protocol), a library was created (see nucleic acid library construction protocol) and sequenced single end 75 bp (see nucleic acid sequencing protocol) Crypts from small intestine were isolated according to a published protocol (Sato 2013). In summary, the small intestine of mouse was washed in ice-cold PBS and opened longitudinally. A microscopy cover slip was used to gently scrape of excess mucus and villus before the intestine was cut into 2-4 mm pieces. The fragments were resuspended in 30 mL ice-cold PBS and pipetted up and down with a 10 mL pipette. Supernatant was discarded and this step was repeated 5-10 times until the supernatant was clear. The fragments were incubated in 2 mM EDTA in PBS for small intestine at 4 C for 30 min with gentle rocking. The supernatant was removed, 20 mL of ice-cold PBS was added and the fragments were pipetted up and down. This washing step was repeated until the crypt fraction appeared as seen in a microscope. The 1-3 consecutive crypt fractions were passed through a 70 um cell strainer and collected into a FCS coated 50 mL tube. The crypts were spun down at 300 xg for 5 min and washed once in 10 mL ice cold PBS at 200 xg for 5 min. Approximately 200-500 crypts were resuspended in 50 uL Matrigel and kept at 4 C. 50 uL of the matrigel solution were added to the center of a pre-warmed 24 well plate and quickly transferred to an incubator at 37 degrees with 5 % CO2. After 5 min the pellet had solidified and 500 uL of basal culture medium was added and the plate was put back into the incubator. Basal culture medium consisted 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 % condition medium (CM) from a cell line producing R-Spondin (kind gift from Calvin Kuo, Stanford University School of Medicine, Stanford, CA, USA) and 10 % Noggin-CM (a kind gift from Hans Clevers, Hubrecht Institute, Utrecht, The Netherlands). The culture medium was replaced every 2-3 days. Organoids were passaged by disrupting them with strong mechanical pipetting, letting the solution cool on ice before centrifuging at 200 xg, 5 min at 4 C and resuspending in matrigel. Organoids were passages 2-3 times before being used in the experiment. References: Sato, Toshiro and Hans Clevers (2013). “Primary Mouse Small Intestinal Epithelial Cell Cultures”. In:Epithelial Cell Culture Protocols: Second Edition. Ed. by Scott H. Randell and M. Leslie Fulcher. Methods in Molecular Biology. Totowa, NJ: Humana Press, pp. 319-328.isbn: 978-1-62703-125-7.doi:10.1007/978-1-62703-125-7_19.527url:https://doi.org/10.1007/978-1-62703-125-7_19. 10 ng/mL cytokine (IL-13 Peprotech #210-13, IL-22 Peprotech #210-22, IFNG BioLegend #575306) was applied to wells 24 hours after splitting and the organoids where then incubated for 24 hours with cytokine. Three 24 well plate wells where combined together per sample for RNA extraction. RNA was extracted with Quick-RNA Microprep Kit according to the manufacturers instructions (Zymo, R1050). Library preparation was done with Lexogen SENSE mRNA kit
ENA-FIRST-PUBLIC
2021-05-01T00:23:35Z
ENA-LAST-UPDATE
2020-06-03T20:14:28Z
External Id
SAMEA6872442
INSDC center name
Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
INSDC first public
2021-05-01T00:23:35Z
INSDC last update
2020-06-03T20:14:28Z
INSDC status
public
Submitter Id
E-MTAB-9182:IL13IFNG_M1_S5
age
7 to 8
broker name
ArrayExpress
cell type
epithelial cell of small intestine
common name
house mouse
developmental stage
adult
disease
normal
genotype
wild type genotype
growth condition
organoid culture
individual
A
organism part
epithelium of small intestine
sample name
E-MTAB-9182:IL13IFNG_M1_S5
scientific_name
Mus musculus
sex
male
strain
C57BL/6