GSM8190843: CD11bhiLy6Chi cells from bone marrow (BM) cells without human mesenchymal stem/stromal cells (MSC) coculture; Mus musculus; RNA-Seq
CD11bmidLy6Cmid cells were isolated from GM-CSF-stimulated BM cells in the presence of MSCs, as were CD11bhiLy6Chi cells from GM-CSF-stimulated BM cells in the absence of MSCs, by staining with anti-CD11b and anti-Ly6C Abs (all from Invitrogen/Thermo Fisher Scientific) in FACS buffer (Invitrogen/Thermo Fisher Scientific) and sorting using a flow cytometer (BD FACSAria™ III Cell Sorter, BD Biosciences, San Jose, CA). eBioscience™eFluor™ 780 dye (Invitrogen/Thermo Fisher Scientific) was added to stained cells for viability assessment. FACS-sorted CD11bmidLy6Cmid and CD11bhiLy6Chi cells were validated for > 70% viability using a Countess 2 Automated Cell Counter (Thermo Fisher Scientific). The cells along with the reverse transcription master mix were loaded onto the 10x Genomics Chromium platform to capture and barcode the cells, generating single-cell Gel Beads-in-Emulsion (GEMs) according to the manufacturer's instructions. Following reverse transcription, cDNAs with both barcodes were amplified, and a library was constructed using the Single Cell 3' Reagent Kit (v2 chemistry) for each sample. These libraries were then sequenced on an Illumina NovaSeq 6000 System (Illumina, San Diego, CA) in the 2 × 150 bp paired-end mode.