RNA-seq of WT, Mbd3-/- and rescue hiPSC during neuroectodermal differentiation and of WT and Mbd3-/- in mEpiSC during neuroectodermal differentiation

Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. In this study, we investigated the structure and function of the nucleosome remodeller and deacetylase (NuRD) complex in human induced pluripotent stem cells (hiPSCs) and in mouse epiblast stem cells (mEpiSCs). We studied gene expression changes at the self-renewal state (0 days) and at 3, 6 and 12 days of neuroectodermal differentiation in WT, Mbd3-/- and rescue hiPSCs and at 2, 4 and 8 days of neuroectodermal differentiation in mEpiSCs. Sequencing libraries were prepared using the NEXTflex Rapid Directional RNA-seq kit (Illumina) or SMARTer® Stranded Total RNA-Seq Kit v2—Pico Input Mammalian (Takara Bio) and sequenced on the Illumina platform at the CRUK Cambridge Institute Genomics Core facility (Cambridge, UK). Illumina sequence files were converted into FASTQ format. The short sequence reads (75 nucleotides) were aligned to the Human reference genome (hg38; http://genome.ucsc.edu/) or to the Mouse reference genome (mm10; http://genome.ucsc.edu/) and assigned to genes using BWA (Li & Durbin, 2009). We used the Subread package (R statistical tool; http://www.r-project.org/) to count aligned reads. Differentially expressed genes were identified using R package edgeR (Chen, Lun, & Smyth, 2016).