An integrated cell barcoding and computational analysis pipeline for scalable analysis of differentiation at single-cell resolution

This study develops a versatile cell multiplexing and data analysis platform to gain knowledge gain into mechanisms of cell differentiation. We engineer a cell barcoding system in human cells enabling multiplexed single-cell RNA sequencing for high throughput perturbation of customisable and diverse experimental conditions. This is coupled with a new computational analysis pipeline that overcomes the limitations of conventional algorithms by using an unsupervised, genome-wide, orthogonal biological reference point to reveal the cell diversity and regulatory networks in the input scRNA-seq data set. We implement this pipeline by engineering transcribed barcodes into induced pluripotent stem cells and multiplex 62 independent experimental conditions comprising eight differentiation time points and nine developmental signalling perturbations in duplicates. We identify and deconstruct the temporal, signalling, and gene regulatory imperatives of iPSC differentiation into cell types of ectoderm, mesoderm, and endoderm lineages. This study provides a cellular and computational pipeline to study cell differentiation applicable to studies in developmental biology, drug discovery, and disease modelling. ### Competing Interest Statement The authors have declared no competing interest.