Gene inductions in response to BCR-crosslinking in TAK1-deficient B cell

BCR-induced gene expression profile in wild-type and B cell-specific TAK1-deficient B cells; to elucidate how TAK1 regulates BCR-mediated proliferative response Keywords: ordered Purified splenic B cells (CD43 negative) were treated with or without anti-IgM (20 micloG/mL) for 4 h. Total RNA was extracted with an RNeasy kit (Qiagen), double-stranded DNA was synthesized from 10 micloG of total RNA with the SuperScript Choice System (Invitrogen) primed with T7-(dT) 24 primer. These cDNAs were used to prepare biotin-labeled cRNA by an in vitro transcription reaction performed using T7 RNA polymerase in the presence of biotinylated-ribonucleotides, according to the manufacturer’s protocol (Enzo Diagnostics). The cRNA product was purified using an RNeasy kit, fragmented, and hybridized to Affymetrix mouse expression array A430.2 microarray chips, according to the manufacturer’s protocol (Affymetrix). The hybridized chips were stained, washed, and scanned with a GeneArray Scanner (Affymetrix).

本数据集涵盖野生型及B细胞特异性TAK1缺陷B细胞中B细胞受体（BCR, B cell receptor）诱导的基因表达谱，旨在阐明TAK1调控BCR介导增殖应答的具体机制。实验所用细胞为纯化的脾脏CD43阴性B细胞，分别以20 μg/mL抗IgM处理或不做处理，培养4小时。采用RNeasy试剂盒（Qiagen）提取总RNA；取10 μg总RNA，以T7-(dT)24引物为引物，通过SuperScript Choice系统（Invitrogen）合成双链cDNA。以所得cDNA为模板，在生物素标记的核糖核苷酸存在下，利用T7 RNA聚合酶进行体外转录反应，制备生物素标记的cRNA，实验操作遵循Enzo Diagnostics的制造商说明书。随后使用RNeasy试剂盒纯化cRNA产物，将其片段化后与Affymetrix小鼠表达芯片A430.2微阵列芯片进行杂交，操作遵循Affymetrix的制造商说明书。杂交完成的芯片经染色、洗涤后，采用GeneArray Scanner（Affymetrix）进行扫描。