title: Single cell analysis of SHH-patterned hESCs
design: Single cell analysis of SHH-patterned hESCs
library_construction_protocol: Cells were lifted using Accutase and spun down at 300xg for 5 minutes before being resuspended in PBS (w/o calcium or magnesium). H9 hESCs were plated at a density of 400,000 cells/cm2 in 96-well plates coated with Geltrex. SynNotch conditions were seeded at 1:1 ratio of senders:receivers. Neural induction medium was prepared on day 0 and contained neurobasal medium (Gibco, 21103049) supplemented with 1% N2 supplement (Gibco, 17502048), 2% B-27 supplement without vitamin A (Gibco, 12587010), 1% GlutaMAX (Gibco, 35050061), 10 μM SB431542 (STEMCELL TECH, 72234), 100 nM LDN193189 (STEMCELL TECH, 72149), and 10 μM Y-27632. WT H9 hESCs were also plated with 200 ng/mL SHH protein (Sonic C25II, R&D Systems, 464-SH). Medium was prepared (excluding Y-27632) and changed daily, and cells were taken out to 11 days in culture. Cells were encapsulated using the PIPseq method. The PIPseq manufacturer's guide was then followed for nucleic acid extraction. The PIPseq T2 kit v3 chemistry was used. The PIPseq manufacturer's guide was then followed for library preparation.