GSM7096425: scRNAseq for Day 4, replicate 2; Mus musculus; RNA-Seq
mESCs were differentiated using the hanging drop method. Trypsinized cells were suspended in differentiation medium (mESC medium without LIF) to a concentration of 50,000 cells/ml. 20 μl drops (~1000 cells) were then placed on the lid of a bacterial plate and the lid was upside down. After 48 h incubation, Embryoid bodies (EBs) formed at the bottom of the drops were collected and placed in the well of a 6-well ultra-low attachment plate with fresh differentiation medium containing 0.5 μM retinoic acid (RA) for up to 10 days, with the medium being changed daily. The EBs were collected at Day 0, 2, 4, and 10 after retinoic acid treatment. For each time point,the cells were first treated with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher) at 37◦C for 10-15 min with pipetting. Single cell suspension was obtained by passing through 37 μM cell strainer (STEMCELL Technologies) twice. After measuring cell concentration, approximately 1 million of cells were centrifuged at 300 rcf for 5 min. Nuclei were isolated by following the protocol provided by 10x Genomics (Nuclei isolation for single cell multiome ATAC + Gene expression sequencing, CG00365, Rev A). The final nuclei concentration was adjusted to 3000 cell/μL in 1X Nuclei Buffer (10x Genomics). The sample was immediately submitted to Stanford Genomics Service Center (SGSC) for single cell sorting using 10x Chromium Controller (target cells: 5000 per replicate, total 2-3 replicates per time point). For scRNA-seq, we followed 10X Genomics library preparation protocol. The EBs were collected at Day 0, Day 2, Day 4, and Day 10. They were first treated with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher) at 37oC for 10-15 min with pipetting. Single cell suspension was obtained by passing through 37 μM cell strainer (STEMCELL Technologies) twice.  After measuring cell concentration, approximately 1 million cells were centrifuged at 300 rcf for 5 min. The cell pellet was washed once with PBS + 0.04% BSA. The final cell concentration was adjusted to 1000 cell/μL in PBS + 0.04% BSA. The sample was then submitted to Stanford Genomics Service Center (SGSC) for single cell sorting using 10X Chromium Controller (target cells: 5000 per replicate, total 2 replicates per time point). The scRNA-seq library was generated using Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, PN-1000268).  For scATAC-seq, we followed 10X Genomics library preparation protocol. The EBs were collected at Day 0, Day 2, Day 4, and Day 10. Single cell suspension was first obtained using the same procedure shown above. After measuring cell concentration, approximately 1 million cells were centrifuged at 300 rcf for 5 min at 4oC. Cells were lysed by incubating in 100 μL chilled Lysis Buffer for 5 min on ice. After wash with 1 mL chilled Wash Buffer, the nuclei were isolate by centrifuged at 500 rcf for 5 min at 4oC. The final nuclei concentration was adjusted to 3000 cell/μL in 1X Nuclei Buffer (10X Genomics). The sample was then submitted to Stanford Genomics Service Center (SGSC) for single cell sorting using 10X Chromium Controller (target cells: 5000 per replicate, total 2 replicates per time point). The scATAC-seq library was generated using Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit (10X Genomics, PN-1000175). 10X Genomics scRNA-seq and scATAC-seq