RNA-seq analysis of bead-purified B cells of 5-week-old TLR9-/- MRL/lpr mice stimulated in vitro with TLR7 agonists (CL097, invivogen) or left unstimulated.

Purpose: The goal of this study was to develop a B-cell specific gene signature to catalog induced genes after short term in vitro TLR7 stimulation of TLR9-/- MRL/lpr B cells. Methods : Bead purified B cells (5M) from three 5-week-old TLR9-/- MRL/lpr mice were stimulated for 4 hours at 10M/ml with TLR7 agonist CL097 (Invivogen) at 5µg/ml or left unstimulated. RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN). Samples were sequenced on an NextSeq500 (Illumina, Inc) with 2 x 75 bp paired-end reads (20 million reads per sample). Results: a B-cell specific gene signature of induced genes after short term in vitro TLR7 stimulation was cataloged. mRNA profiles of TLR9 deficient (TLR9-/-) B cells stimulated in vitro with TLR7 agonists (CL097, Invivogen) or unstimulated, were generated in three replicates, by sequencing on an Illumina NextSeq500 using PE 150 Cycles High Output flowcell.

研究目的：本研究旨在构建B淋巴细胞（B cell）特异性基因特征，以收录TLR9敲除（TLR9-/-）MRL/lpr小鼠B细胞经短期体外Toll样受体7（TLR7）刺激后诱导表达的基因。
实验方法：从3只5周龄的TLR9敲除（TLR9-/-）MRL/lpr小鼠体内磁珠分选纯化得到B细胞（5×10^6个），以10×10^6个细胞/mL的密度接种培养，加入终浓度5μg/mL的TLR7激动剂CL097（Invivogen公司）刺激4小时，同时设置未刺激对照组。采用RNeasy Plus Mini试剂盒（QIAGEN公司）提取总RNA。使用Illumina NextSeq500测序平台进行双端测序，读长为2×75 bp，每个样本测序获得2000万条读段。
实验结果：本研究成功构建并收录了TLR9敲除（TLR9-/-）MRL/lpr小鼠B细胞经短期体外TLR7刺激后诱导表达的B细胞特异性基因特征。本研究通过三次生物学重复，采用Illumina NextSeq500平台的PE 150循环高通量测序流动槽（PE 150 Cycles High Output flowcell），对经TLR7激动剂CL097（Invivogen公司）体外刺激或未刺激的TLR9缺陷型（TLR9-/-）B细胞进行转录组测序，成功获得其mRNA表达谱。