RNA-seq of definitive endoderm differentiation of human embryonic stem cells in early G1 phase of the cell cycle

The experiment was designed to investigate transcriptional dynamics during definitive endoderm differentiation of human embryonic stem cells (hESCs) in early G1 phase of the cell cycle. For this, the FUCCI system was used to sort hESCs in Early G1 (EG1). Samples were taken at 0h (hESCs), 12h, 24h, 36h, 48h, 60h and 72h in triplicates. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and residual genomic DNA was removed using the On-Column DNase I Digestion Set (Sigma-Aldrich) following manufacturer's instructions. The libraries were generated at a library fragment size between 100 bp to 1 kb with Stranded RNAseq Standard with Oligo dT pulldown. All samples were amplified with a standard 10 PCR cycle before sequencing. The samples were multiplexed and analysed by Illumina Hiseq V4 with a paired end read-length PE75. RNA-seq data analysis was performed following recommendations described in Conesa et al. (DOI https://doi.org/10.1186/s13059-016-0881-8).