Illumina NovaSeq 6000 paired end sequencing; RNA-seq of small intestinal organoids treated with IL-13 for 0, 1, 4 8 and 24 hours and with the BMP signaling inhibitor DMH1
RNA-seq of small intestinal organoids treated with IL-13 for 0, 1, 4 8 and 24 hours and with the BMP signaling inhibitor DMH1
RNA was extracted (see nucleic acid extraction protocol) from small intestinal organoids (see growth protocol) treated with cytokines and DMH1 for different time points (see treatment protocol), a library was created (see nucleic acid library construction protocol) and sequenced paired end 150 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. 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. Intestinal organoids were split and seeded on a new plate and lysed after 48 hours of growth. 10 ng/mL of IL-13 (Peprotech #210-13) were applied to wells 1 hour, 4 hours, 8 hours and 24 hours before lysis.  For some samples 5 uM Dorsomorphin homolog 1 (DMH1 Stem cell technologies #73632) was additionally added 24 hours before lysis.  Three 24 well plate wells were 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 NEB Next(R) Ultra(TM) RNA Library Prep Kit