DNBSEQ-G400 paired end sequencing; Single cell RNA-seq of perinatal mouse hematopoietic stem cells
Briefly, livers were crushed and filtered in phosphate buffered saline (PBS) supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich; Darmstadt, Germany) and 2 mM EDTA (Sigma- Aldrich). Antibody staining was performed in the same media for 15-20 minutes at 4°C, after pre-incubation with purified CD16/CD32 antibody to block Fc receptors. Single ED19.5/PD0 liver CD45.2+Lineage-(Ter119, Gr1, B220, CD8a, CD5)Sca1+Kit+(LSK)CD150+CD48- pnHSCs were index-sorted with 100 μm nozzle into 384-well plates loaded with lysis buffer and silicone oil overlays using a FACSAria Fusion cell sorter (BD Biosciences; Franklin Lakes, New Jersey, USA) with an automated cell deposition unit. No extraction was performed. Libraries were constructed using the Smart-seq3xpress protocol.