title: PRMT_m1278
organism: Mus musculus
description: Protocols: Isolation of small intestinal cryptsSmall intestinal crypts were isolated according to a published protocol [1]. Shortly summarized: The small intestine of mus musculus was washed in ice-cold PBS and opened longitudinally. A microscopy cover slip was used to gently scrape of excess mucus and villus. Furthermore, 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 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 degrees C. 50 uL of the matrigel solution where 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 \% medium from a cell line producing R-Spondin (kind gift from Calvin Kuo, Stanford University School of Medicine, Stanford, CA, USA) and 10 \% medium from a cell line producing Noggin (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 degrees C and resuspending in matrigel. Organoids were passaged for some weeks after isolation from small intestine before they were seeded for the experiment. MS023 was added to the medium immediately after seeding. 48 hours after seeding the media was changed (with MS032 for relevant wells). 120 hours after seeding three wells were pooled together, spun down and media removed. The samples where snap-frozen in N2 and resuspended in 300 uL RNA lysis (Zymo Quick-RNA microprep). References[1] Sato, Toshiro and Hans Clevers (2013). “Primary Mouse Small Intestinal Epithelial Cell Cultures”. In:Epithelial CellCulture 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. url:https://doi.org/10.1007/978-1-62703-125-7_19 RNA was extracted with Quick-RNA Microprep Kit according to the manufacturers instructions (Zymo, R1050). RNA was eluted in 25 uL H20 and concentration was adjusted to 50 ng/uL. Library preparation was done with NEB Next(R) Ultra(TM) RNA Library Prep Kit
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-16T17:05:57Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-07-02T11:21:27Z
attributes.tag: External Id
attributes.value: SAMEA7034791
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2020-12-16T17:05:57Z
attributes.tag: INSDC last update
attributes.value: 2020-07-02T11:21:27Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9291:PRMT_m1278
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: epithelial cell of small intestine
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: organoid culture
attributes.tag: individual
attributes.value: B
attributes.tag: organism part
attributes.value: epithelium of small intestine
attributes.tag: sample name
attributes.value: E-MTAB-9291:PRMT_m1278
attributes.tag: scientific_name
attributes.value: Mus musculus