Chromium single cell 3 reagent kit v2

PBL or colon single-cell suspensions from each patient were pooled with equivalent number of live cells and resuspended at 1–2.5 × 10³ cells/µl in 0.04%BSA/PBS, with the addition of 10 µM Y-27632 (MedChem Express) for colon samples. Samples from unique individuals were pooled, and samples from the proximal (Right,R) or distal (Left,L) colon of the same individual were placed in separate pools, so each sample could later be uniquely identified using demuxlet^{21 (link)}. For the primary biopsy UC and VDZ analysis, the two pools were loaded into four wells each of a Chromium Single Cell 3′ v2 Reagent Kit (10X Genomics), with a total of 8 wells (Supplementary Table 2). 1 × 10⁶ cells of both single-cell colon suspension pools were stained with a custom TotalSeq-A panel, (BioLegend) (Supplementary Table 3) according to the manufacturer’s instructions and loaded into two wells. For all experiments, 60,000 cells were loaded per well and processed for single-cell encapsulation and cDNA library generation using the Chromium Single Cell 3′ v2 Reagent Kits (10X Genomics). TotalSeq-A library generation was performed according to manufacturer’s instructions (BioLegend). Libraries were sequenced on an Illumina NovaSeq6000 to obtain 25,000 reads per cell for the gene expression libraries and 10,000 reads per cell for the TotalSeq libraries.