Single-cell RNA-seq of Orbital Connective Tissues

Single-cell suspensions from orbital connective tissues were derived from 1 TAO patient and 1 healthy control as previously described (30 (link)). Single cells were isolated and lysed with collagenase buffer composed of 5 mg/mL collagenase IV and 10 U/mL DNase I at 37°C for 50 minutes (all from Sigma-Aldrich, St. Louis, USA). Subsequently, filtration and red blood cell removal were performed using the MACS Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). RNA was then reverse-transcribed into cDNA libraries using a 10x Genomics Chromium machine following the manufacturer’s instructions. DNA sequencing was performed on a NovaSeq 6000 Sequencing System (Illumina, CA, USA) using a paired-end 150 sequencing mode. The Illumina output was processed using Cell Ranger 3.0.2 (10×Genomics, CA, USA). Cells of sufficient complexity were clustered and t-distributed stochastic neighbor embedding (t-SNE) plots were generated for visualization using the Seurat R package (31 (link)).