RNA-seq analysis of differentially expressed genes as a function of initial cell-population density during lineage-commitment in mouse embryonic stem cells

We examined all transcriptome-level expressions in three initial cell-population densities (862, 1724 and 5172 cells/cm2) in the first two days of differentiation in N2B27. We collected cells in 10-mL tubes and centrifuged them using a pre-cooled centrifuge. We then extracted RNA from each cell-pellet using the PureLink RNA Mini Kit (Ambion, Life Technologies) according to its protocol. We next prepared the cDNA library with the 3′ mRNASeq library preparation kit (Quant-Seq, Lexogen) according to its protocol. We then loaded the cDNA library onto an Illumina MiSeq system using the MiSeq Reagent Kit v3 (Illumina) according to its protocol. We analyzed the resulting RNA-seq data as previously described (Trapnell et al., Nat Protoc 2012). We performed the read alignment using TopHat, read assembly using Cufflinks and analyses of differential gene expression data using Cuffdiff. We used the reference genome for Mus musculus from UCSC (mm10). We performed enrichment analysis of genes based on their FPKM values (e.g., more than 2-fold expressed when two initial population densities are compared) by using GO-terms from PANTHER (Mi et al., Nucl Acids Res 2019) and custom MATLAB script (MathWorks). We visualized results of pre-sorted, Yap1-related genes (LeBlanc et al., Elife 2018; Mugahid et al., Elife 2020; Yu et al., Oncogene 2018; Huh et al., Cells 2019; Zhu et al., Nature Sci Rep 2018; Zhou et al., Int J Mol Sci 2016; Vigneron & Vousden, EMBO J 2012; Kim et al., Cell 2015) into heat maps that displays the normalized expression value (row Z-score) for each gene and each condition. We analyzed a total of 7 samples (see listed below). Each sample is represented by 3 biological replicates that were acquired from 3 independent experiments.