title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of small intestinal organoids treated with the control medium ENR (EGF, Noggin, R-spondin1) in the presence or absence of muscle supernatant
design: RNA-seq of small intestinal organoids treated with the control medium ENR (EGF, Noggin, R-spondin1) in the presence or absence of muscle supernatant
library_construction_protocol: 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.