Single-Cell Multi-omics RNA and ATAC Sequencing of In Vitro Hematoendothelial Differentiation from Mouse Embryonic Stem Cells
Single-Cell Multi-omics RNA and ATAC Sequencing of In Vitro Hematoendothelial Differentiation from Mouse Embryonic Stem Cells
Day 0,2,3,4,5,6,7, and 8 differentiated mouse embryonic stem cells are collected as embryonic bodies, washed with PBS, dissociated with TrypLE Express Enzyme for 5 minutes, and Trypsin-EDTA for 3 minutes at 37°C in intermittent pipetting. After dissociation, samples are inactivated with PBS+10%FCs, go through a filter (40 μm), and spin down at 1000rpm for 5 minutes. The samples were washed three times with PBS containing 10% FCS. Then, DNase Treatment protocol followed by the manufacturer's recommendations. 10X Genomics 'Single Cell Multiome ATAC + Gene Expression v1' and Single Cell 3' v3 chemistery and protocol used as recomended.  The harvested embryonic bodies were washed three times with PBS, and prewarmed TrypLE was added in a 50 ml falcon tube and placed in a 37°C water bath while shaking with intermittent pipetting for disassociation.  Four minutes later, 3 ml Trypsin/EDTA was added, and the dissociation process continued for at least 7 minutes while visually checking the dissociation of the EB. The disassociation buffer was inactivated with PBS+10%FCS, strained with a 40 µM cell strainer to create a single-cell suspension, washed three times, and stained for cell surface marker staining or prepared for scRNA-seq.   mESCs-Medium: DMEM HyClone™ (cytiva SH30081), 15% - Fetal Bovine Serum (FBS) (Capricorn Scientific FBS-12A, CP18-2152), 1X - MEM Non-Essential Amino Acids Solution (100X) (Gibco 11140-035), 2mM - GlutaMAX™ Supplement (Gibco 35050061), 10mM - HyClone™ HEPES Buffer (cytiva SH30237.01), 1X - Penicillin-Streptomycin (100X) (Sigma-Aldrich P0781), 0.1mM - 2-Mercaptoethanol (50 mM) (Gibco 31350010), 1000U.ml -  ESGRO® Recombinant Mouse LIF Protein 10^7 units/ml (Sigma-Aldrich ESG1107).    Differentiation Medium:  IMDM, GlutaMAX™ Supplement (Gibco 31980048), 15% - FBS (Capricorn Scientific FBS-12A, CP18-2152), 1X - Penicillin-Streptomycin (100X) (Sigma-Aldrich P0781), 0.1 mM - 2-Mercaptoethanol (50 mM) (Gibco 31350010), 50 µg/ml - L-Ascorbic acid (Sigma-Aldrich A5960-25G), 150 μg/mL -Transferrin (Roche 10652202001)  The manufacturer's recommended protocol has been followed. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CaG000338) The manufacturer's recommended protocol has been followed. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CaG000338)