Expression analysis of neuronal differentiation of single mutants UBE3A/PHLDA2/SLC22A18 and double mutant PHLDA2/SLC22A18 cells
Expression analysis of neuronal differentiation of single mutants UBE3A/PHLDA2/SLC22A18 and double mutant PHLDA2/SLC22A18 cells
hESCs were grown on feeder layer MEFs combined with hESC medium until 50-60% confluency (small colonies). Cells were washed with PBS and gently dissociated using 0.25% Trypsin Solution A (Biological Industries) for approximately 5 minutes at 37Â°đś. Dissociated colonies were collected and transferred to non-adherent 10 cm dishes and suspended in confluency). The following day, mTeSR medium was replaced with embryoid body (EB) medium which contains same ingredients as standard hESC medium without bFGF (knockout Dulbeccoâs modified Eagleâs medium (DMEM) supplemented with 15% knockout serum replacement (KSR, Thermo Fisher Scientific), 0.1 mM nonessential amino acids, 2mM L-glutamine, 50 đđ đđ -1 streptomycin, 50 đ˘đđđĄđ  đđ-1 penicillin and 0.1 mM Î˛ mercaptoethanol), for 4 days. After 4 days EB medium was supplemented with 0.1 đM Retinoic Acid (RA, Sigma). Cells were grown in suspension for 17 more days before being plated on 10 cm Matrigel-coated dishes. Cells were grown on Matrigel up to 10 more days before they were collected for downstream analysis. Medium was changed every other day throughout the experiment. For sample collection, cells were washed with PBS and trypsinized. Qiagen RNeasy Kit KAPA Stranded mRNA-Seq Kit