Mission shrna

pLKO.1/puromycin plasmids encoding Mission shRNA (Sigma) sequences were used (table 4 in Supplementary Information). Additionally, an shRNA sequence targeting the 3′ UTR of ADAM15: FWD 5′-CCGGGTTGGACGGGATTGAGGAACTCGAGTTCCTCAATCCCGTCCAACTTTTTG-3′ REV 5′-AATTCAAAAAGTTGGACGGGATTGAGGAACTCGAGTTCCTCAATCCCGTCCAAC-3′, was cloned into pLKO.1 using AgeI and EcorRI restriction sites. The positive clones were selected by XhoI digest according to Sigma Mission shRNA protocol. Lentiviral particles were produced according to Sigma Mission shRNA protocol. Stable downregulation of Claudin-1 or ADAM15 was achieved by 1 µg/mL puromycin selection.

For the shRNA knockdown experiment, an equal number of APC^KO organoids were transduced with lentiviral shRNA constructs against either control (SHC002, MISSION shRNA, Merck,) or BCL-XL (TRCN0000033500, MISSION shRNA, Merck) by spin transduction. Briefly, organoids were collected and trypsinized using TrypLE Express (Thermo Fischer Scientific) for 3 min at 37 °C. Following dissociation, cells were washed, counted, and seeded into 48-well plates in organoid medium (see above) containing 10 µM ROCK inhibitor (Sigma-Aldrich), 8 µg/ml polybrene (Sigma-Aldrich), and 50 µL concentrated virus (AMICON Ultra-15 100k filters, Merck, Schiphol-Rijk, The Netherlands). Plates were spun down at 32 °C for 1 h at 1800 rpm. After ON incubation, cells were collected by washing with PBS and spun down and either processed for RNA extraction or seeded into Matrigel (Corning) for measuring outgrowth. APC^KO organoids were transduced with lentiviral pHEFTIR-EV (empty vector) or pHEFTIR-BCL-XL (overexpressor) constructs [16 (link)] and selected by sorting for the RFP positive population. APC^KO organoids were transduced with lentiviral microRNA inhibitors (Merck) targeting hsa-miR-17-5p (HLTUD0264), hsa-miR18a-3p (HLTUD0292), and a negative control cel-mir-243-3p (HLTUD002C). Transduced cells were selected with 4 µg/ml puromycin (InvivoGen, Toulouse, France) for 7 days.

To knockdown CXCR4 in GL26-Cit cells, pLKO.1-puro lentiviral plasmids encoding four different shRNA hairpins constructs specific for mouse CXCR4 (NM_009911) along with puromycin resistance cassette were purchased from Sigma Aldrich as part of the mission shRNA Consortium. Each shRNA clone was tested for its' ability to knockdown CXCR4 expression. Second-generation lentiviruses encoding two shRNA constructs (TRCN0000028704, CXCR4 shRNA1 and TRCN0000028749, CXCR4 shRNA2) were prepared according to manufacturer's instructions (Clonetech_LentiXTX transfection kit). (Cat# 631317). 5 x106 HEK293TX cells were seeded 24 hours prior to transfection with each of the shRNA construct along with Lenti-X HTX packaging mix, then cells were incubated for 48 hours at 37°C. Lentiviral particles were purified from culture supernatant. GL26-Cit cells were infected with purified mouse CXCR4–specific shRNA lentivirus for 48-72 hours and subjected to puromycin selection for 2 weeks. A western blot analysis was performed to validate stable CXCR4 knockdown in GL26-Cit cell line. Similar methodology was used to create GL26-Cit-NT control cell-line, which consisted pLKO.1-puro lentiviral expression vector and non-targeting shRNA hairpin construct (mission shRNA, Cat#:SHC002, Sigma-Aldrich).