HAND1 ChIP-seq at day 3 of cardiac differentiation from human embryonic stem cells
HAND1 ChIP-seq at day 3 of cardiac differentiation from human embryonic stem cells
Embryoid bodies (EBs) were dissociated into single cells with TryPLE and fixed with 1% formaldehyde (Pierce) for 10 minutes at room temperature, then quenched with 0.125 M Glycine for 5 minutes at room temperature. Cells were centrifuged and washed three times with cold PBS, then the pellets snap-frozen in a dry ice/ethanol bath and stored at -80°C until required.  Differentiations were performed in serum-free BPEL medium (Ng et al., 2008) containing 1 μg/ml insulin using an embryoid body (EB) system. EBs were formed by depositing 2500 cells in 50 μl differentiation medium per well in V-bottomed 96-well plates (Greiner). The following growth factors were present for the first 3 days of differentiation: 25 ng/ml BMP4 (R&D Systems), 25 ng/ml Activin A (Miltenyi Biotech) and 1.65 μM CHIR99021 (Selleckchem). 5 μM of the ALK5 inhibitor SB431542 (Generon) (or equivalent volume of the vehicle-only (DMSO) control), was added on day 2 as a 2 μL bolus and mixed.  Pellets were thawed on ice and re-suspended in Sonication/IP buffer (140 mM NaCl, 50 mM HEPES pH 7.5, 1 mM EDTA pH 8.0, 1%, 0.1% SDS, 0.1% sodium deoxcycholate, 1X protease inhibitor). 3 x 106 cells in 150 µl per TPX 1.5 ml tube (Diagenode) was sonicated for 40 cycles (30 seconds on/30 seconds off on High Power) using a Bioruptor (Diagenode). Libraries were prepared using MicroPlex Library Preparation Kit v3 (Diagenode)