title: Illumina HiSeq 2500 sequencing; scRNA-seq of Multi-lineage differentiating Stress Enduring (Muse) cell and non-Muse cells isolated from bone marrow-derived mesenchymal stem cells
design: scRNA-seq of Multi-lineage differentiating Stress Enduring (Muse) cell and non-Muse cells isolated from bone marrow-derived mesenchymal stem cells
library_construction_protocol: Bone marrow derived mesenchymal stem cells (BM-MSCs) were purchased from Lonza (Basel, Switzerland). Cells were cultured at 37°C, 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA, USA) with 10% FBS (HyClone, Logan, UT, USA), 1ng/ml basic fibroblast growth factor (b-FGF; Miltenyi Biotec, Bergisch Gladbach, Germany), 0.1 mg/ml kanamycin (Invitrogen), and 1% GlutaMAX (Invitrogen). Human Muse cells and non-Muse MSCs were isolated by fluorescence-activated cell sorting as described previously (Kuroda et al., PNAS, 2013). Briefly, BM-MSCs were incubated with rat anti-stage specific embryonic antigen (SSEA)-3 antibody (1:1000; BioLegend, San Dige, CA, USA)  for one hour at 4°C. After three-times washes, cells were further incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rat IgM (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for one hour at 4°C. SSEA-3(+) cells were collected as Muse cells and SSEA-3(-) cells as non-Muse MSCs using FACSAria II cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). RNA extraction was prepared using Chromium single cell 3' Kit v3 (10x Genomics, Pleasanton, CA, USA)  Sequencing library was prepared using Chromium single cell 3' Kit v3 (10x Genomics, Pleasanton, CA, USA)