title: iPS2-CITE-seq with arrayed polyclonal hiPSC targeting of either SMAD2 or B2M
library_construction_protocol: Digestion with versene and trippleE. Then cells were labeled with ADT 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) against B2M and TotalSeq™-B antibodies for multiplexing before loading on a reaction of 3' Next GEM single cellv3.1 kit. Since antibodies were used for both multiplexing hashing and CITE-seq in this experiment, the 10X reference protocol CG00149 Rev was followed with minor adjustments. 1×10^6 live cells per population were first incubated for 30 minutes at 4°C with TotalSeq™-B0057 antibody and washed once with PBS and 1% BSA. Then, CTR-treated samples were incubated with TotalSeq -B0251 hashtag, while TET-treated samples were incubated for 30 minutes at 4°C with TotalSeq -B0252 hashtag and washed three times with PBS and 1% BSA. After three washes, cells were incubated for 15 minutes at room temperature with Fixable Viability Dye eFluor™ 780 (Invitrogen 1:1000 dilution). Live cells were 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 CG000317 RevE (with feature technology for surface protein). Cells were loaded on a chip G. The single-cell gene expression and ADT libraries were generated using the Chromium Next GEM Single Cell 3' v3.1 workflow (CG000317 RevE). GEM formation, reverse transcription (RT), and post-RT cleanup were performed by following the user guide. cDNA amplification, using Feature cDNA primers 2, was performed for 11 cycles. A Sample Index PCR with indexes TT (gene expression library) and NN (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 for the gene expression library or 1.2x single-sided size selection for the ADT library. The iPS2seq library was constructed from the amplified cDNA (6.34 ng input) as described in the supplementary protocol 3 section. The first iPS2-seq PCR amplification was performed for 16 cycles, then, after 1.8x SPRIselect purification, the PCR was diluted to ~0.1 ng/µL and indexed with dual-indexes TT. Final purification was again performed using a 0.7x–0.9x SPRIselect size selection. All libraries (GEX, iPS2seq, and ADT) were quantified by Tapestation and Qubit dsDNA HS assays. 