GSM8844220: parathyroid, normal, rep1; Rattus norvegicus; RNA-Seq
The excised glands were minced and enzymatically digested at 37°C for 40 minutes with gentle shaking in a solution containing TL (1 mg/mL, Roche), Dispase (2 mg/mL, Miltenyi Biotec), and DNase I (0.05 mg/mL, Sigma). Every 20 minutes, dissociated cells were collected using a 10 mL plastic pipette containing fetal bovine serum (FBS). The resulting cell suspension was filtered through a 20 μm mesh filter (BD Biosciences), centrifuged at 300 g for 5 min, and incubated in red blood cell lysis buffer (155 mM NH₄Cl, 0.1 mM Na₂EDTA, 10 mM KHCO₃) for 3 minutes. The dissociated parathyroid cells were then resuspended and stored in Dulbecco's phosphate-buffered saline (DPBS). Single-cell cDNA library preparation and 3' single-cell RNA sequencing were conducted by CapitalBio Technology (Beijing, China). Libraries were prepared using the 10x Genomics Chromium platform, following the manufacturer's instructions with the Single Cell 3' Library and Gel Bead Kit V3 (10x Genomics, 1000075) and the Chromium Single Cell B Chip Kit (10x Genomics, 1000074). Cells were suspended in PBS containing 0.04% BSA, and approximately 8,000 cells were loaded onto the Chromium Single Cell Controller (10x Genomics) to generate single-cell gel bead emulsions (GEMs). Captured cells were lysed, and released RNA was barcoded through reverse transcription within individual GEMs. RT-PCR was performed using an S1000™ Touch Thermal Cycler (Bio-Rad) under the following conditions: 53°C for 45 min, 85°C for 5 min, followed by a hold at 4°C. cDNA was recovered, amplified, and quality-assessed using the Agilent 4200 system (conducted by Beijing Cade Biotechnology Co., Ltd.). Single-cell RNA-seq libraries were constructed using the Single Cell 3' Library and Gel Bead Kit V3, following the manufacturer's protocol. Finally, sequencing was performed on an Illumina NovaSeq 6000 platform, generating paired-end 150 bp (PE150) reads with a minimum sequencing depth of 100,000 reads per cell (conducted by Beijing Cade Biotechnology Co., Ltd.).