title: GSM8484535: Old; Mus musculus; RNA-Seq
library_construction_protocol: Three aged mice and three young mice were sacrificed by cervical dislocation. And then detachd the femur from the tibia. Cut the proximal and distal ends of the bones, the bone marrow was flushed from mouse tibias and femurs using a 25G × 5/8" needle and plastic syringe and then kept in phosphate buffer saline (PBS) with 5% fetal bovine serum (FBS) on ice. Bone marrow cells were centrifuged at 400×g at 4℃ for 10 min. Resuspend the cells in 2 ml NH4Cl2 (pH: 7.2-7.6) for 5 min at room temperature (RT) to lyse the erythrocytes. Then centrifuge for 10 min, at 400×g, at 4℃ and resuspend the cells in 1 ml of 5% PBS/FBS. For fluorescence-activated cellsorting, cells were blocked with anti-mouse CD16/32 (BioLegend, 101302) on ice for at least 10min. Cells were then incubated for 30 min at 4℃with preconjugated fluorescence-labeled antibodies with the following combinations: APC anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody (BioLegend, 108412), FITC anti-mouse/human CD11b Antibody (BioLegend, 101206). Fixable Viability Dye eFluor™ 450 (eBioscience, 65-0863) was used to stain cells to assay for viability. Cells were sorted with BD FACSAria III and the selected populations were isolated for next experiments. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). 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 10x 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.