scRNA-seq of Bcl9/9l fl/fl;Rosa26-CreERT2 small intestinal organoids
scRNA-seq of Bcl9/9l fl/fl;Rosa26-CreERT2 small intestinal organoids
Small intestinal organoids were harvested with ice-cold medium and transferred to a 15ml tube. Ice cold Advanced DMEM/F12 was added till 10ml, and the organoids were pelleted at 4°C for 5min at 400g. 'Matrigel cloud' was carefully removed and organoids were pelleted again in 10ml ice cold advanced DMEM/F12. Organoids were resuspended in 1ml TrypLE express enzyme, Phenol Red (12605010, Thermo Scientific) and incubated in a 37°C waterbath till the organoids were fully dissociated to single cells while gently shaking and occasional pipetting with a p1000 to prevent clumping. Cells were checked under a brighfield microscope every 5 min to assess dissociation. Once the organoids were fully dissociated to single cells the digestion was stopped with 500ul BCS, immediately topped till 10ml with ice cold Advanced DMEM/F12 and spin at 4°C for 5 min at 500g. The cells recovered from each condition were counted (range 100-200k) and scored for successful dissociation to single cells.  Mouse small intestinal organoids were established as previously described in Sato et al. 2007. Crypts were isolated from the entire length of the small intestine of one Bcl9fl/fl;Bcl9lfl/fl;Rosa26CreERT2(HET) animal to establish the organoid line.  Organoids were culture in 15ul BME (3434-005-02, Trevigen) droplets in culture medium containing advanced DMEM/F12 (12634010, ThermoFisher Scientific) supplemented with 100U/ml Penicillin/Streptomycin (15276355, ThermoFisher Scientific), 2mM Glutamax (11574466, ThermoFisher Scientific), 10mM HEPES (15630-056, ThermoFisher Scientific), and 1.25mM N-acetylCysteine (A9165-5G, Sigma Aldrich), freshly added mEGF (50ng/ml, 315-09, PeproTech), 2% Noggin-Fc conditioned media (N002-100ml, AVSbio), 2% Rspo3-Fc conditioned media (R001 – 100ml, AVSbio) at 37°C and 5% CO2. For passaging, cell culture medium was removed and BME was broken into small pieces by scraping, followed by vigorous pipetting with ice-cold advanced DMEM/F12. Crypts were centrifuged at 400g for 5 min at 4°C. The supernatant was carefully removed, the pellet was resuspended in 50:50 advanced DMEM/F12:BME and plated on pre-warmed culture plates. Medium was refreshed every other day, and organoids were split once a week in a 1:3 ratio. . Organoids were grown untill day 5 and treated with 1μM 4-OHT (H7904-5MG, Sigma-Aldrich) for 24 hours to delete Bcl9/9l. Organoids were harvest at day 8 for qPCR or scRNA-seq. Nucleic acids were extracted from fixed nuclei according to the manufacturer's protocol (EXW02050, Parse Biosciences). The recovered cells were fixed according to the manufacturer's protocol (ECW02050, Parse BioSciences) including 0.5% BSA in the fixation solution (15260037, Fisher Scientific) and stored at -80°C. Before barcoding, cells suspensions were thawed and recounted and loaded on the first barcoding plate ac-cording to the manufacturer's WT mega v2 sample loading table aiming at ~10k cells per condition. Further barcoding and sequencing library generation was performed according to the manufacturer's protocol, generating 6 sub-libraries, with a target of 20000 cells each (note that more experiments were mixed in the sequencing library, amounting to ~1500 cells / condition / library). 