GSM5620836: hiPSC_day+20_2; Homo sapiens; RNA-Seq
Single cell analysis of differentiating iPSCs and mouse embryos cells was performed using inDrops as previously reported (Klein et al.). UCP1-mCherry iPSC line was differentiated into brown adipocytes as described above. Cultures were harvested on day 6, 10, 15, 20, 30 of the primary differentiation and day 20 and 40 after replating.  The cultures were washed with PBS and dissociated into single cells using 2.5mg/ml Collagenase and 0.05% Trypsine EDTA in PBS for 5-15 minutes at 37ºC. Dissociated cells were run through 70µm cell strainer followed by 30µm cell strainer, spun at 300g for 5 minutes and resuspended in DMEM supplemented with 5% fetal bovine serum. Cells were again spun at 300g for 5 minutes and resuspended in DMEM + 5% fetal bovine serum, this was repeated 2-3 times to remove debris. Final resuspension was made in PBS + 0.1% bovine serum albumin. Cell density was adjusted to 200,000 cells/ml. For day 6, 10, 15 and 20, 2500 cells were collected from each sample. For samples 30 day, 20 day after replating and 40 day after replating, at least 3000 cells were collected from each sample from two independent differentiations. For analyzing mouse embryonic tissues, embryos were collected on embryonic day 13.5, 14.5 and 15.5 from wildtype CD1 IGS mice. For each stage, back tissue dorsal to rib cage at the level of forelimb was dissected out. Neural tube/spinal cord and dorsal root ganglion were removed wherever possible. For E15.5, epidermis and underlying dermis was removed before dissociating the tissue. Dissected tissues were washed several times in Hanks' Balanced Salt Solution. Tissues were transferred to microfuge tube with 200µl of 2.5mg/ml Collagenase and 0.05% Trypsine EDTA in PBS, chopped into small pieces using scissors and incubated at 37ºC for 15 minutes with intermittent shaking. Tissues were mechanically dissociated by triturating several times using wide-bore 1ml pipette. The resulting cell suspension was mixed with HBBS + 10% FBS, filtered through a 30µm cell strainer and spun at 300g for 5 minutes. To remove red blood cells, cell pellet was resuspended and incubated with RBC Lysis Solution for 5 minutes at room temperature. Cells were washed with HBSS + 10% FBS for 2-3 times to remove debris. Final suspension was made in PBS + 0.1% BSA. Cell density was adjusted to 200,000/ml. For each stage, cells were collected from two littermate embryos. For E13.5-14.5  embryo, 3000 cells were encapsulated and sequenced from each embryo. For E15.5, 5000 cells were collected and sequenced from each embryo. Single-cell RNA-Seq libraries were prepared using a protocol customized for the inDrops platform (see Zilionis et al., Nature Protocols 2017)