title: iPS2-10X-seq experiment on specific clones targeting with an shRNA either SMAD2 or B2M
library_construction_protocol: Digestion with versene and trippleE. Then cells were labeled with ADT, CMO, and sorted for vital cells and then loaded on the 10X controller. We processed the organoids with trypsin and obtained a single-cell suspension. We then labeled the cells with TotalSeq™-B0057 antibody (BioLegend) and cell multiplexing oligos (CMO) before loading on a reaction of 3' Next GEM single cellv3.1 kit. Cells were first incubated with antibodies and then with CMOs to combine CMOs and antibodies. Antibody labeling was performed as by following 10x Genomics demonstrated protocol CG00149 Rev D. 1×106 live cells per population were stained with TotalSeq™-B0057 antibody in 100 µL final volume in PBS and 1% BSA. Following a 30-minute incubation at 4°C, cells were washed with PBS and 1% BSA (Miltenyi) and then labeled with CMOs following the 10x Genomics protocol (CG000391 RevA). After three washes with PBS and 1% BSA, cells were incubated for 15 minutes at room temperature with Fixable Viability Dye eFluor™ 780 (Invitrogen 1:1000 dilution). Cells were washed again and sorted with a SONY SH800S sorter with a 100 µm chip in FACS tubes, then pooled together in equal quantities, and resuspended at 1,600 cells/µL to be loaded on the 10X chromium with a target of 16,000 cells per reaction. 10X genomics technology with CG000390 RevC (with cell multiplexing oligos and B2M antibody staining Totalseq-B). Cells were loaded on a chip G. The single-cell gene expression, multiplexing, and antibody-derived tag (ADT) libraries were generated using the Chromium Next GEM Single Cell 3' v3.1 workflow (CG000390 RevC with cell multiplexing oligos and B2M antibody staining Totalseq-B). GEM formation, reverse transcription (RT), and post-RT cleanup were performed by following the user guide. cDNA amplification used with Feature Barcode Primer 3 and was performed for 11 cycles. A Sample Index PCR with indexes TT (gene expression library), NN (CMO library), and NT (ADT library) was performed as suggested by the 10X guidelines. All libraries were purified using SPRIselect beads with 0.7x–0.9x double-sided size selection. hIPS2-seq barcode enrichment protocol was performed with custom primers binding on the TET repressor. All libraries (GEX, CMO, and ADT) were quantified by Tapestation and Qubit dsDNA HS assays. 