GSM7405524: SCG_12k_run66; Mus musculus; RNA-Seq
Adult C57BL/6 males were sacrificed at 24 weeks of age and sympathetic ganglia (SCG and Stellate) were quickly extracted under a stereomicroscope. Tissue was digested for 30 min with collagenase (2.5 mg/ml) and DNase (5 U/ml) in HBSS at 37 ◦C, washed and further digested with trypsin (0.25%) for 30 min at 37◦C with shaking. The samples were mechanically triturated, and the cell suspension was then filtered through a 40-μm cell strainer and centrifuged at 400 g for 5 min. After aspiration of the supernatant, the pellet was resuspended in DMEM media with 10% FBS and 1 vol. of 40% w/v iodixanol solution. The cell suspension was carefully overlayed with an Optiprep gradient composed of 3 ml 22% w/v iodixanol solution and 0.5 ml DMEM and centrifuged at 800 g for 25 min. The viable cells were carefully collected from the top interface and the cell suspension was concentrated to 300 µl by removing the supernatant. The cell suspension (approximately 10,000 cells per channel) was then loaded onto the single cell 3' chip and placed on a 10X Genomics chromium controller instrument to generate single cell gel beads in emulsion (GEMs). Single cell RNA-seq libraries were prepared using the chromium single cell 3' library & cell bead kit according to the manufacturer's protocol. Libraries were sequenced with an Illumina NextSeq500 platform to a depth of approximately 300 million reads per library with 2 × 50 read length