Scgm medium

Peripheral blood mononuclear cells (PBMCs) were isolated from unfractionated peripheral blood by Ficoll-Hypaque density gradient (density 1.077 g/ml, Biochrom, VWR) centrifugation. For functional and phenotypic analyses, PBMCs were resuspended in SCGM medium (CellGenix) containing 10% human serum (pooled human AB serum, # P041702, Pan-Biotech) and cultured for 2 days without or with 400 U/ml of IL-2 (kindly provided by the NIH, USA).

Ten thousand CD34⁺ cells from CB and mPB were cultured in SCGM medium (Cellgenix) with the following recombinant hematopoietic cytokines: recombinant human stem cell factor (rhSCF) (100 ng/ml), recombinant human thrombopoietin (rhTPO) (100 ng/ml) and recombinant human fms‐related tyrosine kinase‐3 ligand (rhFlt3‐L) (100 ng/ml). Cells were cultured in 24‐well tissue culture plates at 37°C in an atmosphere of 5% CO₂ and treated with candidate small molecules NVP‐BEZ235 (10 nM), cucurbitacin I (100 nM), fenretinide (100 nM) and calmidazolium (500 nM). Medium were changed every two days and candidate small molecules were added.

The fresh CD34⁺ cells were applied to cell culture in vitro or to single-cell RNA-seq (scRNA-seq). CD34⁺ cells were resuspended in SCGM medium (Cellgenix) using the following recombinant hematopoietic cytokines: recombinant human stem cell factor (rhSCF) 100 ng/ml, recombinant human thrombopoietin (rhTPO) 100 ng/ml, recombinant human fms-related tyrosine kinase-3 ligand (rhFlt3-L) 100 ng/ml and cultured in 24-well tissue culture plates at 37 °C in an atmosphere of 5% CO₂ for 48 h (Thermo Fisher). The DNBelab C4 platform was used to perform scRNA-seq. Single-cell suspensions were used for droplet generation, demulsification, microbead collection, reverse transcription, and cDNA amplification to generate barcode libraries. The manufacturer’s protocol was used to construct indexed libraries. QubitTM ssDNA Assay Kit (Thermo Fisher Scientific; #Q10212) was used to quantify the sequencing libraries. DNA nanoballs (DNBs) are loaded into a pattern nanoarray and sequenced at ultra-high throughput with the following read lengths used by the DIPSEQ T1 sequencer: 30 bp for read 1, inclusive of 10 bp cell barcode 1, 10 bp cell barcode 2 and 10 bp unique molecular identifier (UMI), 100 bp of transcript sequence for read 2 and 10 bp for sample index.

Fresh CD34⁺ cells were immediately cultured ex vivo or used for single‐cell RNA‐seq (scRNA‐seq). For cell culture, CD34⁺ cells were resuspended in SCGM medium (Cellgenix) with the following recombinant hematopoietic cytokines: recombinant human stem cell factor (rhSCF) 100 ng/ml, recombinant human thrombopoietin (rhTPO) 100 ng/ml, recombinant human fms‐related tyrosine kinase‐3 ligand (rhFlt3‐L) 100 ng/ml and cultured in 24‐well tissue culture plates at 37°C in an atmosphere of 5% CO₂ for 48 h (Thermo Fisher). Then, cells were collected for scRNA‐seq. scRNA‐seq were performed by the DNBelab C4 platform.²⁶ In brief, single‐cell suspensions were used for droplet generation, emulsion breakage, beads collection, reverse transcription and cDNA amplification to generate barcoded libraries. Indexed libraries were constructed according to the manufacturer's protocol. The sequencing libraries were quantified by QubitTM ssDNA Assay Kit (Thermo Fisher Scientific; #Q10212). DNA nanoballs (DNBs) were loaded into the patterned nano arrays and sequenced on the ultra‐high‐throughput DIPSEQ T1 sequencer using the following read length: 30 bp for read 1, inclusive of 10 bp cell barcode 1, 10 bp cell barcode 2 and 10 bp unique molecular identifier (UMI), 100 bp of transcript sequence for read 2 and 10 bp for sample index.