Cord blood processing
Umbilical cord blood from anonymous healthy donors was obtained through the National Cord Blood Program from New York Blood Center. Within 48 hours after cord blood collection, mononuclear cells (MNCs) were isolated from each cord blood unit by density centrifugation using Ficoll-Paque PREMIUM (GE Healthcare #17-5442-03), and enriched for CD34+ cells using MACS separation (Miltenyi Biotec #130-100-453). Briefly, umbilical cord blood was diluted two folds using DPBS without calcium and magnesium (Corningļ›š #20-031-CV), layered on top of 15 ml Ficoll-Paque PREMIUM in a 50 ml Falcon tube, and spun down at 850 g for 30 min at room temperature with the brake off. The mononuclear cell layer was then isolated and washed with MACS buffer (DPBS with 0.5% BSA, Sigma-Aldrich #A8806-5G) after red blood cell lysing with ACK lysing buffer (Life Technologies #A10492-01). MNCs were then enriched for CD34+ cells by incubating them with magnetic beads conjugated to mouse anti-human CD34 antibody for 30 min, and passed through a magnetic MACS LS column (Miltenyi Biotec # 130-042-401). CD34+ cells would bind to the LS column and later flushed off and collected in 5 ml MACS buffer. For Drop-seq experiments, two consecutive enrichment steps were performed to increase the purity of enriched CD34+ cells.