hPSC Differentiation into SAPs

Utilizing a modified dual-SMAD inhibition differentiation protocol developed by the Studer laboratory at the Memorial Sloan Kettering Cancer Center, we performed in vitro differentiations of hPSCs into SAPs. Over the course of a 40-day differentiation, cells were cultured and sorted on day 16 for the CD49d maker (SOX10 positive cells), when cells are committed to trunc neural crest cells. Cells were harvested at the neural crest and hSAP stages. RNA was isolated from the collected cell pellets by lysing the cells in TRIzol Reagent (ThermoFisher catalog #15596018) and inducing phase separation with chloroform. Subsequently, RNA was precipitated with isopropanol and linear acrylamide and washed with 75% ethanol. The samples were resuspended in RNase-free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 534–850 ng of total RNA with DV200% varying from 38–74% was used for ribosomal depletion and library preparation using the TruSeq Stranded Total RNA LT Kit (Illumina catalog #RS-122-1202) according to manufacturer’s instructions with 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a 50 bp/50 bp paired end run, using the HiSeq 3000 / 4000 SBS Kit (Illumina). On average, 48 million paired reads were generated per sample and 35% of the data mapped to the transcriptome.