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. 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.

细胞特异性转录应答在批量RNA测序（bulk RNA sequencing）的均值分析中丢失。
我们针对雄性C57BL/6小鼠的冷冻肝脏样本开展了单细胞核RNA测序（single nuclei RNA sequencing, 简称snSeq），以探究其对2,3,7,8-四氯二苯并对二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD）的应答反应。在对照组与处理组的16015个测序细胞核中，共检测到约19907个肝脏表达基因。共鉴定出11种细胞（亚）类型，其中包含特征明确的肝细胞亚群，这与肝脏的细胞多样性特征相符。TCDD处理使巨噬细胞占比从0.5%提升至24.7%，而中性粒细胞仅在处理组样本中存在。差异表达基因的数量与芳香烃受体（Ahr）的基础表达水平相关。除各细胞（亚）类型中预期的功能富集结果外，RAS信号通路在非实质细胞中呈现显著富集。单细胞核RNA测序还鉴定出一类高表达Gpnmb的库普弗细胞亚群，该结果与膳食诱导的非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）模型特征一致。总体而言，单细胞核RNA测序可区分细胞特异性转录变化与细胞群比例变化，这些变化与TCDD的肝毒性效应相符。
出生后第28天的雄性C57BL/6小鼠（n=2）经口灌胃给予芝麻油溶剂对照，或按30 μg/kg的剂量给予TCDD，每4天给药1次，持续28天。于给药第28天（即出生后第52天，PND 52）迅速采集肝脏样本，经液氮速冻后保存于-80℃。从冷冻肝脏中分离细胞核，经DAPI染色后通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）制备单细胞核悬液，随后将其加载至10X基因组学（10X Genomics）Single Cell 3' V3测序平台。