Transcriptomic Profiling of Temperature-Induced Cell Differentiation

Both differentiated (39°C) and undifferentiated (37°C) cells were trypsinized, and scRNA-seq libraries were generated following the Chromium 10X pipeline[78 (link)]. Four replicates at each temperature across >17,000 cells were assayed. Cell capture, cDNA generation, and library preparation were performed with the standard protocol for the Chromium Single Cell 3’ V3 reagent kit. Libraries were quantified with the Qubit dsDNA High Sensitivity Assay (Invitrogen) in combination with the High Sensitivity DNA Assay (Agilent) on the Agilent 2100 Bioanalyzer. Single-cell RNA-sequencing libraries were pooled and sequenced on an Illumina NovaSeq 6000 (SP flow cells), using 2×50 bp reads per library, to a combined depth of 1.6 billion reads. The quality of sequencing was evaluated via FastQC. Paired-end reads were aligned to the mouse reference genome (mm10) using the CellRanger v3.0.1 pipeline. Unique molecular identifier (UMI) counts were quantified per gene per cell (“cellranger count”) and aggregated (“cellranger aggr”) across samples with no normalization.