GSM4502449: hiPSC_PAX7_2; Homo sapiens; RNA-Seq
For generating cell suspensions from NCRM1 human iPSC derived PAX7-Venus positive cells, PAX7-Venus reporter line was differentiated as described (Chal et al., 2016). On day 27, cells were dissociated and captured using inDrops technique. For dissociation, cells were incubated in 2.5mg/ml Type IV Collagenase and 0.05% Trypsin EDTA for 15 minutes at 37 ºC. After mechanical dissociation using a wide bore pipette, cells were added to 5% FBS in DMEM and passed through a 70mm nylon Cell Strainer, spun down at 300g for 5 min and resuspended in 0.1% FBS in PBS. Cells were filtered through a 30mm filter and Venus expressing cells were collected using flow cytometry. Cell density was quantified manually and was adjusted to 150,000 cells/ml for encapsulation. The dissociation method and flow cytometry were optimized to achieve >90% viable cells and minimize cell doublets. Cells were collected from two independent experiments. Library construction was performed according to custom inDrops protocol as in Zilionis et al. Nature Protocols 2017. Single-cell RNA-Seq libraries were prepared using a protocol customized for the inDrops platform (see Zilionis et al., Nature Protocols 2017)