title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of Dand5 KO and WT mESC
library_construction_protocol: The cells were lysed directly on the culture plate using Tri Reagent (Sigma). After addition of the reagent, cell lysates were homogenised using a pipette and immediately used for RNA isolation. Samples were taken at day 0, day 5, day 6, day 8 and day 10. Undifferentiated Dand5 KO and the well-characterized and widespread E14 mESC lines were used to test cells pluripotency potential and spontaneous differentiation by the hanging droplet method. Briefly, cells were dissociated into a single cell suspension and resuspended in fresh mESC medium without LIF. Then, mESCs were cultured in hanging drops (500 cells per droplet) for 48 hours until the formation of embryoid bodies (EBs).  Then, the EBs were cultured in static suspension until day 5, followed by adherent culture in 0.1% gelatin-coated wells up to day 10, prompting spontaneous differentiation. The culture medium was renewed every day. Total RNA was extracted from mouse mESCs and EBs, at several days of differentiation, using TRI Reagent® (Sigma) and the Direct-zolTM RNA MiniPrep Kit (Zymo Research) according to the manufacturer's instructions. Libraries were constructed using Stranded mRNA Library Prep Kit.