title: Illumina NovaSeq 6000 sequencing; scRNAseq for bone marrow cells from Cebpαcre/+ R26EYFP/+ SbdsF/F (neutropenia) mice vs Cebpαcre/+ R26EYFP/+ Sbds+/+ (Ctrl) mice
design: scRNAseq for bone marrow cells from Cebpαcre/+ R26EYFP/+ SbdsF/F (neutropenia) mice vs Cebpαcre/+ R26EYFP/+ Sbds+/+ (Ctrl) mice
library_construction_protocol: Bones were crushed in FACS buffer (PBS+0.5%FCS) with sterilized mortar and pestle. BM cells were pelleted by centrifuging and passed through a 40µM filter. RBCs in BM were lysed in ACK lysing buffer. BM was next divided in two parts. The first part was directly stained with differentiated cell markers and subsequently  B and T Cells (B220+, CD3+), NK Cells and a myeloid rest fraction (B220-, CD3-, NKp46- and NK1.1-) were separately sorted. The second part of the BM was lineage depleted by magnetic-activated cell sorting (MACS) according to manufacturer's protocol (130-090-858, Miltenyi Biotec) and subsequently stained with HSPC markers. HSPC subsets (HSC+MPP (LKS CD48-), HPC1 (LKS CD48+CD150-)) were then sorted separately.  BF cells were prepared by incubating bone fragments after crushing in 0.25% Collagenase Type I (07902, STEMCELL Technologies) for 45 min in 37°C water bath, with vortexing every 15 min, washed in 20mL FACS buffer and passed through a 40µM filter again.  Bone fraction was lineage- and CD45 depleted by adding biotinylated anti-CD45.1 and .2 antibodies (respectively A20, 104, Biolegend) to the above mentioned MACS protocol. Subsequent niche staining enabled the sort of various populations: endothelial cells (CD31+), mesenchymal stromal cells(MSC) (CD31-,Sca1+,CD51+), osteolineage cells (OLC)(CD31-,Sca1-,CD51+).  In order to enrich for rare populations all subsets mentioned above were sorted separately into Dulbecco's Modified Eagle Medium (DMEM)+10% FCS, relatively enriched, pooled together and then encapsulated for barcoding and cDNA synthesis using the Chromium Single-cell 3′ Reagent kit v3 (10x Genomics) The sorted cells were encapsulated for barcoding and cDNA synthesis using the Chromium Single-cell 3′ Reagent kit v3 (10x Genomics) 3' gene expression library was constructed according to the manufacturer's recommendations. The quality and quantity of libraries was assessed with an Agilent 2100 Bioanalyzer with 2100 Expert version B.02.11.SI811 software and a High Sensitivity DNA kit