GSM8182210: PBMC_Aged+AA; Mus musculus; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated from C57BL/6 mice by collecting 1-2 ml of peripheral blood in EDTA-coated tubes, diluting it in an equal volume of RPMI 1640 medium, and layering it over lymphocyte separation medium in centrifuge tubes. After centrifugation at 400 g for 25 minutes, the intermediate white layer containing PBMCs was collected, centrifuged at 500 g for 10 minutes, and half of the supernatant was discarded. The cells were subsequently rinsed with 2 ml of RPMI 1640 medium and subjected to centrifugation at 500 g for 5 minutes. The resulting supernatant was removed, and the cell pellet was reconstituted in 500 µL of fetal bovine serum (FBS) (Gibco). Cell viability was assessed by staining with 0.4% trypan blue (Merck Millipore) and examining under a microscope, with only cells demonstrating over 80% viability being utilized for library preparation. The single-cell libraries were prepared utilizing the Chromium™ Controller and the Chromium™ Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Pleasanton, CA). Single cells, reagents, and gel beads containing 10X Barcodes were enclosed within Gel Bead-In-EMulsions (GEMs), facilitating single-cell lysis and reverse transcription. Subsequent to GEM disruption, cDNA underwent amplification through PCR, with subsequent size selection (200-300 bp). The fragments were subjected to end-repair, A-tailing at the 3' ends, and adapter ligation. The adapter-ligated fragments underwent purification utilizing double-sided Solid Phase Reversible Immobilization (SPRI) beads. Following sample index PCR, a subsequent round of SPRI purification was carried out. The fragment distribution of the prepared library was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and its concentration was quantified via real-time quantitative PCR employing TaqMan probes. Subsequent sequencing was conducted on an Illumina platform.