Impact of Hells loss-of-function on DNA methylation in B cells during humoral immune responses: kinetic comparison of Mb1-Hells knockout and control mice by bulk RNA sequencing.

The transcriptomic impact of Hells loss-of-function was quantified by RNA sequencing of sample from conditional knockout (Mb1-Cre+/WT HellsF/F) and littermate control mice (Mb1-CreWT/WT HellsF/F). Three time points were chosen to follow the kinetic: day 0 for naive B cells collection, day 7 and day 14 post NP-CGG intraperitoneal immunization for germinal center B cells collection. At each time point, 4 mice of each genotype were used. Live cells were FACS sorted (AriaIII) using surface markers as follows: B220+CD21+CD23+ for naive B cells, and B220+CD95+GL7+ for germinal center B cells. Due to their low frequency, germinal center B cells were FACS-sorted twice using the same gating strategy. Collected samples were lysed and processed for RNA extraction.本研究通过对条件性敲除小鼠（Mb1-Cre+/WT HellsF/F）及其同窝对照小鼠（Mb1-CreWT/WT HellsF/F）的样本开展RNA测序（RNA sequencing），定量解析了Hells基因功能丧失（Hells loss-of-function）的转录组学效应。本研究选取三个时间点以追踪实验动力学进程：第0天用于收集初始B细胞（naive B cells），NP-CGG腹腔免疫后第7天及第14天用于收集生发中心B细胞（germinal center B cells）。每个时间点下，每种基因型均使用4只小鼠。活细胞借助荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS，型号AriaIII）完成分选，分选所用表面标记如下：初始B细胞采用B220+CD21+CD23+标记，生发中心B细胞采用B220+CD95+GL7+标记。鉴于生发中心B细胞的细胞丰度较低，需采用相同的门控策略（gating strategy）进行两轮FACS分选。收集得到的样本经裂解后，进行RNA提取。