RNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

Pluripotent stem cells can differentiate into all embryonic germ layers, a process that is orchestrated by the dissolution of pluripotency network and activation of pro-differentiation pathways. Yet, the genes essential for these cell fate transitions in human are still elusive. Here, we used a genome-wide loss-of-function library established in haploid hPSCs and differentiated these cells into into the three germ layers. We analyzed the transcriptome of the differentiated mutant library cells to assess their differentiation status, as well as to determine lineage-specific expression of essential genes for the differentiation of hESCs into each germ layer. hESCs were differentiated towards definitive endoderm, early mesoderm and neuroectoderm. For each germ layer, three biological replicates of differentiated and undifferentiated cells were analyzed.