HSPC subpopulations were isolated using FACS as previously described.15 (link) For flow cytometric analysis of intracellular clusterin (CLU) and LGALS9 expression, the IntraPrep Leukocytic Permeabilization Reagent Kit (Beckman Coulter) was used. Dead cells were excluded in the analysis using Zombie Aqua Fixable Viability Stain (BioLegend). A list of all antibodies is provided in supplemental Table 2.
For MS analysis, 25 000 HSC/MPPs and CMP/MEPs were sorted into protein low-binding micro-centrifuge tubes (Eppendorf) and prepared for MS analysis. Details on sample preparation are provided in the supplemental Material. For RNA sequencing (RNA-seq) analysis, up to 10 000 HSC/MPPs, CMP/MEPs, CMPs, GMPs, and MEPs were sorted into RNeasy lysis buffer (Qiagen) containing β-mercaptoethanol. For the probes labelled PV1/8/12, CON1, CON2, and CON3, samples from different individuals had to be pooled to guarantee adequate HSC/MPP numbers for downstream MS and RNA-seq measurements (supplemental Table 1).
For MS analysis, 25 000 HSC/MPPs and CMP/MEPs were sorted into protein low-binding micro-centrifuge tubes (Eppendorf) and prepared for MS analysis. Details on sample preparation are provided in the supplemental Material. For RNA sequencing (RNA-seq) analysis, up to 10 000 HSC/MPPs, CMP/MEPs, CMPs, GMPs, and MEPs were sorted into RNeasy lysis buffer (Qiagen) containing β-mercaptoethanol. For the probes labelled PV1/8/12, CON1, CON2, and CON3, samples from different individuals had to be pooled to guarantee adequate HSC/MPP numbers for downstream MS and RNA-seq measurements (supplemental Table 1).
