BM-MNC samples were thawed, washed and incubated for 30 minutes on ice with Hoechst 33258 staining dye (Cat #94403; Sigma Aldrich, Gillingham, UK) and the following antibodies (all at 1:100): anti-CD34-APC (clone 581; #555824; BD Biosciences, Oxford, UK); anti-CD38-PE (HIT2; #25-0389-42; eBioscience, Altrincham, UK); anti-Lin-FITC (pooled antibodies targeting CD2/3/4/7/8/10/11b/14/19/20/56/235a; #25-0389-71; BD Biosciences;). Viable Lin-CD34+CD38- cells were sorted using a FACS Aria III sorter (BD Biosciences). To ensure maximum purity with minimal contamination of the targeted population, sorted samples were immediately subjected to a second sort (when cell numbers permitted) using identical gating. Up to 2,000 Lin-CD34+CD38- cells were targeted per sample, based on the empirically-determined 10X capture efficiency rate (40-50%).  Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf
Experimental Factor: disease
normal