Single cell RNA-seq analysis of young (2 months) and old (18 months) mouse hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSCs) represent a rare population of cells residing in the Bone Marrow (BM) at the top of hematopoietic hierarchy. A critical balance is maintained between self-renewal and lineage differentiation of HSCs to maintain hematopoietic homeostasis. With aging, this balance is altered with an increase of self-renewal long term HSCs and a myeloid biased differentiation, which favors the appearance of myeloid leukemias and anemias. This experiment aims to understand molecular mechanisms that cause this aged-related disequilibrium in the mouse. To this end, we generated single cell RNA-seq data from pools of young and old hematopoietic stem and progenitor cells (HSPCs), isolated from mouse BMs. Overall design: Mice at young age (2 months) or at old age (17-18 months) were sacrified and bone marrow (BM) harvested. Pools of 2 or 3 BMs of the age-matched mice were formed to obtain 2 samples of young mice (young_A and young_B) and 2 samples of old mice (old_A and old_B). For each sample, cells were lineage depleted by using the Lineage Cell Depletion Kit (Miltenyi) and labelled with the following antibody cocktail: anti-cKit, anti Sca-1 anti CD34, anti CD150, anti CD48 and anti Flt3 antibodies to purify Lin-Sca1+cKit+ Flt3- cells (HSPCs) by multi-parameter fluorescence-activated cell sorting (FACS). We used the 10x Genomics platform and FACS purified HSPCs were loaded 30 min after the sorting onto a Chromium Single Cell Chip and processed with the Chromium Controller (10x Genomics) according to the manufacturer's instructions for single cell barcoding at a target capture rate of 4000 individual cells per sample. Libraries were prepared using Chromium Single-Cell 3' Reagent Kits v2 (10x Genomics) and were sequenced using an Illumina NextSeq500 sequencer to an average depth of about 45,000 reads per cell for Young_A and Old_B samples -and about 130,000 reads per cell for Old_A and Young_B samples.