The culture, differentiation, and sorting of embryonic stem cells (ESCs) in vitro

The data of mESCs hematopoietic differentiation and RNA-seq derived from the publications³⁰. The hematopoietic differentiation of hESCs was performed using STEMdiff Hematopoietic Kit (STEMCELL Technologies; 05310) following the instructions. Fluorescence-activated cell sorting (FACS) was performed according to the following two schemes, first based on the differentiation time point including day3, day6, day9 and day 12. Second, cells were sorted by the following markers: CD309-Percp/cy5.5 CD31-APC CD34-PE, CD43-APC and CD45- FITC (all kits Biolegend). The total RNA of these FACS-sorted cells was extracted into RNA by using Trizol reagent (Life Technologies; 15596026), and 1 μg RNA was reverse-transcribed into complementary DNA (cDNA) using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) according to the manufacturer’s instructions (Vazyme; R223-01).