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
#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.