RNA sequencing on sorted viable Zsg-positive and Zsg-negative populations

To genetically characterize lPBNcold neurons, we used RNA sequencing combined with a fluorescence-activated cell sorter (FACS). Prepared single cell suspension was sorted based on green fluorescence (indicative of cold responsiveness) and DAPI (indicates cell integrity and viability) through FACS, yielding a 79.34% of viable cells and 5.88% of Zsg-positive cells among singlet gated counts. To determine differentially expressed genes, we performed RNA sequencing on sorted viable Zsg-positive and Zsg-negative populations.