Single-Cell Roadmap of Early Hemato-Endothelial Development: Roles of Atf3, Zfp711, And Bcl6b
Single-Cell Roadmap of Early Hemato-Endothelial Development: Roles of Atf3, Zfp711, And Bcl6b
Day 7 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 37C in intermittent pipetting. After dissociation, samples are inactivated with PBS+10%FCs, go through a filter (40 um), and spin down 1000rpm for 5 minutes. The samples were washed three times with PBS+10%FCS. Than, 10X Genomics 3' CellPlex Kit Set A (CMO sample multiplexing) protocol followed and continued with  Chromium Next GEM Single Cell 3ʹReagent Kits v3.1. 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)  Chromium Next GEM Single Cell 3' Reagent Kits v3.1 protocol has been followed. Chromium Next GEM Single Cell 3' Reagent Kits v3.1 protocol has been followed with dual indexing library construction.