Amicon ultra 15 100k filters

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.

Purified SARS2/SNFPP+CMP+TEV-H8STREPH6 was subjected to size-exclusion chromatography using a superose 6 increase 10/300 GL column (Cytiva, Tokyo, Japan) in 1 × PBS buffer. The peak fractions were collected and concentrated by ultrafiltration using Amicon ultra-15 100K filters (Merck, USA). Thyroglobulin (669 kDa), Ferritin (440 kDa), and Aldolase (158 kDa) were used as molecular weight markers.