title: GSM6050661: Early periodontitis [Ligature_Day1]; Mus musculus; RNA-Seq
library_construction_protocol: At 1 days after ligature placement, the gingival tissue from the ligature side of the palate was harvested (n=4). Gingival tissues were cut into 1-mm pieces and placed in digestion buffer containing 1 mg/ml collagenase II (Life Technologies, Grand Island, NY), 10 units/ml DNase I (Sigma-Aldrich) and 1% bovine serum albumin (BSA; Sigma-Aldrich) in Dulbecco's modified Eagle's medium (DMEM; Life Technologies) for 20 min at 37°C on a 150 rpm shaker. The tissues were passed through a 70µm cell strainer. The collected cells were pelleted at 1,500 rpm for 10 min at 4°C and resuspended in phosphate buffered saline (PBS; Life Technologies) that was supplemented with 0.04% BSA (Cell suspension A).  The remaining tissues were further incubated in 0.25% trypsin (Life Technologies) and 10 units/ml DNase I for 30 min at 37 ℃ on a 150 rpm shaker. The combined trypsin-released cells and collagenase II-released cells were counted and the equal number of cells (2,000~3,000) from each animal were combined in the group for single cell RNA sequencing (10X Genomics, San Francisco, CA).