nucleic acid sequencing protocol
Samples were sequenced on an Illumina HiSeq2000 machine with a 50 cycle single-read method and a target insert size of 175bp.
Definitive endoderm was generated by treating H9 embryonic stem cells with Activin A for 3 days as previously described by Spence, et al. Nature 470: 105-109 (2010) and McCracken, et al. Nature Protocols 6:1920-1928 (2011).
nucleic acid library construction protocol
RNA was extracted from samples and then prepared for RNAseq using the Illumina TruSeq mRNA method
H9 embryonic stem cells were differentiated into definitive endoderm and then treated with FGF4/Chiron (a Wnt agonist) in order to generate intestinal organoids as previously described by Spence, et al. Nature 470: 105-109 (2010) and McCracken, et al. Nature Protocols 6:1920-1928 (2011).
Cells were cultured in mTeSR on matrigel coated plates and collected for RNA when ~70% confluent with no signs of spontaneous differentiation.