GSM8499352: control, replicate1, scRNAseq; Mus musculus; RNA-Seq
Freshly harvested whole bone marrow (BM) cells from Map4k4-cKO or control mice were isolated using ice-cold Hank's Balanced Salt Solution without Mg2+/Ca2+ (HBSSwo), supplemented with 10 mM HEPES at pH 7.2. Subsequently, the whole BM cells from control or Map4k4-cKO mice were respectively divided into two aliquots. One aliquot underwent treatment with red blood cell lysis buffer to remove erythrocytes, while the other was subjected to enrichment for c-Kit+ BM hematopoietic stem and progenitor cells (HSPCs) using magnetic bead separation. The isolated c-Kit+ BM HSPCs were then artificially mixed with the corresponding BM cells at a ratio of 2:3 to establish a combined c-Kit/BM cell population. Library was performed according to the manufacter's instructions (single cell 3' protocol, BMKMANU DG1000). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the DG1000 Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added