STING gain-of-function disrupts lymph node organogenesis and innate lymphoid cell differentiation in mice. Bennion et al

Heterozygous STING N153S mutant mice lack lymph nodes and Peyer’s patches. Lymph node development requires a RORT+ lymphoid tissue inducer (LTi) cells. In STING N153S fetuses, there are fewer LTi cells. These LTi cells arise in the fetal liver from CD127+ 47+ progenitor cells. We hypothesized that STING N153S impairs LTi cell survival or function, and that transcriptional abnormalities might be detectable by single-cell RNA-sequencing.

To enrich for this rare cell type, single Lin–CD45+cKITintCD127+47+ cells were sorted directly into a 96-well plate (one cell per well) containing 2 l of 10x lysis buffer (Takara catalog no. 635013) and 5% RNAse inhibitor (Promega catalog no. PRN2611). After sorting, plates were immediately frozen at -80 degrees Celsius. Single-cell RNA sequencing of each well was performed at the Genome Technology Access Center (GTAC) at Washington University in St. Louis. A total of 48 WT and 48 STING N153S cells were sequenced.

异源STING N153S突变小鼠缺乏淋巴结和派尔氏集合淋巴结。淋巴结发育需要RORT+淋巴组织诱导（LTi）细胞。在STING N153S胎儿中，LTi细胞数量较少。这些LTi细胞起源于CD127+ 47+祖细胞所在的胎儿肝脏。我们假设STING N153S损害了LTi细胞的存活或功能，转录异常可能通过单细胞RNA测序被检测到。为了富集这种稀有的细胞类型，单Lin–CD45+cKITintCD127+47+细胞被直接分选至一个包含2微升10x裂解缓冲液（Takara产品目录号635013）和5%RNA酶抑制剂的96孔板（每孔一个细胞）。分选后，板立即在-80摄氏度下冷冻。每个孔的单细胞RNA测序在圣路易斯华盛顿大学的基因组技术访问中心（GTAC）进行。总共对48个野生型和48个STING N153S细胞进行了测序。