H1-AAVS1-TetOn-dCas9-KRAB hESCs were split to single cells with
TrypLE Express (Thermo, 12604). Cells were resuspended in mTeSR1
supplemented with 10 μM Y27632 (Tocris, 1254) and 500 ng/mL
doxycycline. 2 × 106 cells were plated into each well of a
6-well plate pre-coated with Growth Factor Reduced Matrigel (Corning,
356231). On Day 1, cells were fed with mTeSR1. On Day 2, cells were fed with
RPMI1640 (Thermo, 21870) supplemented with 0.2% Hyclone FBS (GE Healthcare,
SH30070.03), 100 ng/mL Activin A (R&D Systems, 338-AC-01M), 3 μM
CHIR 99021 (Tocris, 4423), and 50 nM PI 103 (Tocris, 2930). On Days 3 and 4,
cells were fed with RPMI1640 supplemented with 0.2% Hyclone FBS, 100 ng/mL
Activin A, and 250 nM LDN-193189 (Tocris, 6053). Media was changed every 24
hours. For perturbation experiments and CRISPRi screening, the media was
supplemented with 500 ng/mL doxycycline daily.
TrypLE Express (Thermo, 12604). Cells were resuspended in mTeSR1
supplemented with 10 μM Y27632 (Tocris, 1254) and 500 ng/mL
doxycycline. 2 × 106 cells were plated into each well of a
6-well plate pre-coated with Growth Factor Reduced Matrigel (Corning,
356231). On Day 1, cells were fed with mTeSR1. On Day 2, cells were fed with
RPMI1640 (Thermo, 21870) supplemented with 0.2% Hyclone FBS (GE Healthcare,
SH30070.03), 100 ng/mL Activin A (R&D Systems, 338-AC-01M), 3 μM
CHIR 99021 (Tocris, 4423), and 50 nM PI 103 (Tocris, 2930). On Days 3 and 4,
cells were fed with RPMI1640 supplemented with 0.2% Hyclone FBS, 100 ng/mL
Activin A, and 250 nM LDN-193189 (Tocris, 6053). Media was changed every 24
hours. For perturbation experiments and CRISPRi screening, the media was
supplemented with 500 ng/mL doxycycline daily.