Comparison manual and two types of bioprinted kidney organoids by single cell RNA-seq

We compared kidney organoids generated manually ('Man') to those generated by bioprinting single cell deposition ('R0') and thin bioprinted lines ('R40'). 4 independent pools of iPS cells were differentiated (Takasato et al, 2015; Howden et al, 2019) and used to generate kidney organoids. Each pool was used to generate each of the 3 types of conformations: Manual organoids, bioprinted organoids composed of a single deposition of cells (R0) and bioprinted organoids composed a thin line of cells (R40). R0 and R40 were composed of 110k starting cells, while Manual was 230k. Each pool of cells was used to set up all 3 conditions, giving a total of 4 replicates per condition. Organoids were dissociated after 12 days of growth and each replicate was antibody labelled using a hash-tag oligo (HTO) barcode (BioLegend). Barcoded cells were pooled by conformation to generate and used to generate 3 10x single cell libraries. Each library represents a single conformation (Manual, R0, R40) and contains the 4 barcoded replicates. HTO sequences were separated by SPRI fractionation and used to geneate a separate HTO library. Standard 10x mRNA libraries and HTO libraries were sequenced separately, mapped and counted to give a count matrix then combined in Seurat for final analysis. Methods are described in detail in a paper under review, which will be referenced here once published.