GSM7437897: PHZ_BM_LSK; Mus musculus; RNA-Seq
Bone marrow cells were isolated by crushing the long bones, pelvic bones, vertebrae, and sternum with mortar and pestle in HBSS without calcium and magnesium, supplemented with 2% heat-inactivated bovine serum. Splenocytes were dissociated by smashing the entire spleen between slides. Cells were triturated and filtered through a 40 μm cell strainer to obtain a single-cell suspension. For isolation of LSK cells, cells were incubated with fluorescent-conjugated antibodies and pre-enriched by selecting c-kit+ cells using paramagnetic microbeads and autoMACS (Miltenyi Biotec). Nonviable cells were excluded from sorts and analyses using the viability dye DAPI. FACS was performed with FACSAria II (BD Biosciences). The library was prepared following the manufacturer's instructions (Chromium Next GEM Single Cell 3' Kit v3.1, 10x Genomics). In brief, sorted LSK cells were resuspended in the master mix and loaded into the chip, along with partitioning oil and gel beads, to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained within each individual GEM was reverse-transcribed to cDNA, which included an Illumina Read 1 primer sequence, Unique Molecular Identifier, and the 10x Barcode. The pooled barcoded cDNA was then cleaned up using Silane DynaBeads, amplified by PCR, and appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During library construction, the Illumina Read 2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added.