Bulk ATAC-seq analysis of human H1 ESCs differentiated into definitive endoderm and foregut endoderm with our without 2uM Retinoic Acid.

To examine the effect of a high pulse of Retinoic Acid (2uM) during a 24 hour temporal window (days5-6) on the accessible chromatin status of human H1 ESCs differentiated into foregut endoderm. Overall design: Differentiation of definitive endoderm and foregut endoderm from human H1 ESCs: For differentiation, hESCs were plated as single cells in a Matrigel-coated 24-well dish using accutase (Stem Cell Technologies), at a density of 200,000 cells per well in mTesR1 with ROCK inhibitor Y-27632 (10uM; Stemgent). On the following day, hESCs were differentiated to DE as follows: cells were treated with Activin A (100ng/mL; Cell Guidance Systems) for three days (72hours) in RPMI 1640 media (Invitrogen) containing increasing concentrations of defined fetal bovine serum (dFBS; Invitrogen): 0% (day1), 0.2% (day2), and 2.0% (day3 onwards). In addition, BMP4 (50 ng/mL; R&D Systems) was added for 24 hours on the first day of DE induction. For foregut endoderm differentiation, cells were cultured in RPMI 1640 media with 2.0% dFBS and Noggin (200 ng/mL R&D Systems) for 72 hours (days4-6). Retinoic acid (RA; 2 µM; Sigma Aldrich) was added for 24 hours from day5-6 to promote posterior foregut fate. Replicate wells were harvested for bulk ATAC-seq analysis after 72 hours (end of day 3) or further differentiated for 72 hours into foregut endoderm, with or without Retinoic Acid treatment for 24 hours (day5-6) and harvested at end of day 6 for bulk ATAC-seq.