Application of singe nuclei RNA sequencing to assess the hepatic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Cell-specific transcriptional responses are lost in the averages of bulk RNA sequencing. We performed single nuclei RNA sequencing (snSeq) on frozen liver samples from male C57BL/6 mice in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Approximately 19,907 hepatic genes were detected across 16,015 sequenced nuclei from control and treated samples. Eleven cell-(sub)types were identified including distinct hepatocyte sub-populations, consistent with the cell diversity of the liver. TCDD increased macrophages from 0.5% to 24.7%, while neutrophils were only present in treated samples. The number of differentially expressed genes correlated with the basal expression level of Ahr. In addition to expected functional enrichments within each cell-(sub)type, RAS signaling was enriched in nonparenchymal cells. snSeq also identified a Kupffer cell subtype highly expressing Gpnmb, consistent with a dietary NASH model. Overall, snSeq distinguished cell-specific transcriptional changes and population shifts consistent with the hepatotoxicity of TCDD. Overall design: Male C57BL/6 mice aged postnatal day 28 mice (N = 2) were orally gavaged with sesame oil vehicle or 30 µg/kg TCDD every 4 days for 28 days. On day 28 (PND 52) livers were immediately collected, frozen in liquid nitrogen, and stored at -80°C. Nuclei were isolated from frozen livers, stained with DAPI, and subjected to FACS in order to generate a single nuclei suspension which was loaded onto the 10X Genomics Single Cell 3' V3 platform.