GSM8464577: acupuncture, replicate3, scRNAseq; Mus musculus; RNA-Seq
Mice were anesthetized and perfused with 10 mL of pre-chilled phosphate-buffered saline (PBS) via the left heart ventricle. Tissues from the ST36 acupoint were excised and stored in cold 1640 medium . Then, the tissues were minced on ice using small scissors and incubated in 5 mL of a digestion buffer containing 0.25 mg/mL Liberase TH Research Grade enzyme and 50 μg/mL DNase I at 37°C for 30 min, with gentle shaking twice during incubation. Digestion was halted using 5% fetal bovine serum. The digested tissue was then passed through a 70-μm cell strainer into pre-chilled PBS twice on ice. Cells were pelleted by centrifugation at 400 g at 4°C for 5 min. Following centrifugation, the cell pellet was incubated with 3 mL of red blood cell lysis buffer  on ice for 5 min and pelleted again by centrifugation at 400 g at 4°C for 5 min. Subsequently, the cells were washed, re-suspended in pre-chilled PBS twice, and filtered through a 40-μm cell strainer to remove debris or cell aggregates. Finally, the cells were centrifuged at 400 g at 4°C for 5 min, re-suspended in 500 μL of pre-chilled PBS with 0.04% bovine serum albumin, and dead cells were removed using Dead Cell Removal Micro Beads . Libraries were prepared according to Illumina's instructions accompanying the Illumina NovaSeq 6000