GSM6605116: adult male mouse bone-marrow cells short-read 10x scRNA-seq, replicate 2 library 2; Mus musculus; RNA-Seq
Femurs and tibias of the euthanized animals were dissected using sterile surgical techniques and transferred to a petri dish containing the basal medium (Minimal Essential Medium supplemented with 10% FBS and 1X antibiotics antimycotic solution (Gibco)). The dorsal and distal ends of femurs and tibias were cut with scissors to reveal the bone marrow cavity, and the bone marrow cells were flushed out with the basal medium using needles/syringes. Cells were then passed through 100 μm and 40 μm cell strainers, washed, pelleted, and resuspended in 5 mL of ACK lysis buffer (Lonza BP10-548E, Basel, Switzerland) for 5 minutes at room temperature. After determining their density and viability, cells were resuspended at 1 million cells per ml of 0.04% BSA/PBS prior to generating the 10× GEM single-cell libraries. Single cells were captured in droplet emulsion using the Chromium Controller (10× Genomics, Pleasanton, CA), and GEM single-cell libraries were constructed according to the 10× Genomics protocol using the Chromium Single-Cell 30 Gel Bead and Library 3' V3 kit (10× Genomics, Pleasanton, CA). In brief, cell suspensions were diluted in PBS with 0.04% BSA to a final concentration of 1×106 cells/mL (1,000 cells per μL). Cells were loaded in each channel with a target output of 10,000 cells per sample. All reactions were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) with a 96 Deep Well Reaction Module. Twelve cycles were used for cDNA amplification and sample index PCR. Amplified cDNA and final libraries were evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip. paired-end ilimina next-seq