title: Sample 2
organism: Homo sapiens
description: Protocols: hESCs (H9 from WiCell) were grown in defined culture conditions as described previously (Brons et al, 2017). Pluripotent H9 cells were passaged weekly using collagenase IV and maintained in chemically defined medium (CDM) supplemented with Activin A (10 ng/ml) and FGF2 (12 ng/ml). Cells were passaged every 4-6 days with collagenase IV as clumps of 50-100 cells, and dispensed at a density of 100-150 clumps/cm2. The culture media was replaced 48 hours after the split and then every 24 hours. The generation of FUCCI-hESC lines has been described in Pauklin and Vallier (2013) and are based on the FUCCI system described in Sakaue-Sawano et al. (2009). Differentiation into endoderm was performed for up to 72 hours with a combination of cytokines as described in Pauklin and Vallier (2013) and Pauklin et al. (2016). For cells sorted by FACS, the cells were collected and immediately placed into the endoderm differentiation media. Endoderm specification was performed in CDM with polyvynilic acid (CDM-PVA) prepared without insulin and supplemented with 50ng/ml FGF2, 1µM Ly-294002 (Promega), 100ng/ml Activin A, and 10ng/ml BMP4 (R&D). 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 for RNA-sequencing were generated by the Wellcome Sanger Institute Illumina Bespoke Sequencing Facility and sequencing was performed onsite. The libraries were generated at a library fragment size between 100 bp to 1 kb with Stranded RNAseq Standard with Oligo dT pulldown.