Single cell RNA-seq and VDJ-seq of B cells from sheep red blood cells-primed WT and Stch cKO mice

Once activated, B cells produce the antibodies needed to fight against infection. B cells are necessary for adaptive or specific immunity, which focuses on the destruction of foreign invaders that have gotten past the bodies initial defenses. Heat shock protein family is a group of highly conserved molecular chaperones with important functions in protein folding such as antibody and in signal transduction. Stch (Heat shock 70 kDa protein 13) is a member of the heat shock protein 70 family and is found associated with microsomes. Members of this protein family play a role in the processing of cytosolic and secretory proteins. To explore the role of Stch in B-cell activation and antibody production, we evaluated the transcriptional events and the role of stch pathway on the activation and antibody production of B cells by single cell RNAseq. Splenic lymphocytes were isolated from 7-9-week-old female Stchf/f wildtype (WT) mice and B-cell specific Stch knocked-out (CD19creStchf/f) mice (3 mice/group) using Ficoll (Ficoll-Paque Plus, GE Healthcre). B cells were sorted using B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany). Their transcriptomes were captured using a 10x chromium system.B细胞一经激活，便可产生抵御感染所需的抗体。B细胞是适应性免疫（adaptive immunity）与特异性免疫（specific immunity）的必要组成部分，这类免疫靶向清除突破机体初始防御屏障的外来入侵者。热休克蛋白家族（Heat shock protein family）是一类高度保守的分子伴侣（molecular chaperones），在蛋白质折叠（包括抗体折叠）及信号转导过程中发挥重要功能。Stch即热休克蛋白70kDa蛋白13（Heat shock 70 kDa protein 13），隶属于热休克蛋白70家族，且已被发现与微粒体（microsomes）相关联。该蛋白家族的成员参与胞质蛋白与分泌蛋白的加工过程。为探究Stch在B细胞活化与抗体生成中的作用，本研究通过单细胞RNA测序（single cell RNAseq）分析转录事件，并评估了Stch通路对B细胞活化及抗体生成的影响。本研究采用Ficoll-Paque Plus分层液（GE Healthcare），从7~9周龄雌性Stchf/f野生型（wildtype, WT）小鼠及B细胞特异性Stch敲除（CD19creStchf/f）小鼠（每组3只）中分离脾淋巴细胞；使用B220磁珠（货号：130-049-501，德国美天旎生物技术Miltenyi Biotec）分选B细胞，并通过10× Chromium系统完成其转录组捕获。