Targeted RNA-Seq of Human Naïve CD4+ cells

Purpose: The purpose of this RNA-seq experiment was to perform a correlation analysis of mRNA expression levels in naïve CD4+ T cells and LPS-stimulated monocytes from patients with an IKZF1 mutant haploinsufficient phenotype (H167R-a and H167R-b are two siblings carrying IKZF1 p.H167R mutation) versus patients with an IKZF1 mutant dominant negative phenotype (C1, G1) along with five healthy normal controls (HC). Method: Targeted RNA-seq library preparation was carried out using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies) which is designed to profile over 20,000 distinct human RefSeq genes using a highly multiplexed amplification method. Each amplicon represents a unique targeted gene (one transcript per gene). The average size of each amplicon is ~150 bp. For library preparation, each sample was run in duplicate and a cDNA library was first generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified using Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified using Agilent Bioanalyzer high sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with four individual samples per pool. Pooled libraries were amplified and enriched using the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies), using Ion PI HiQ kit and chip version 3.

实验目的：本RNA测序（RNA-seq）实验旨在开展相关性分析，对比携带IKZF1突变单倍体不足表型的患者（H167R-a与H167R-b为两名携带IKZF1 p.H167R突变的同胞）、携带IKZF1突变显性负性表型的患者（C1、G1），以及5名健康对照（HC）的初始CD4+ T细胞与脂多糖（LPS）刺激单核细胞的mRNA表达水平。实验方法：采用Ion AmpliSeq转录组人类基因表达试剂盒（Life Technologies）进行靶向RNA-seq文库制备，该试剂盒通过高度多重扩增策略，可对超过20000个独立人类RefSeq基因进行表达谱分析。每个扩增子对应一个唯一的靶向基因（每个基因对应一条转录本），平均扩增子长度约150 bp。文库制备流程如下：每份样本设置两次重复，首先从至少10 ng总RNA中合成cDNA文库；通过Ion AmpliSeq技术对cDNA进行条码标记与扩增；随后采用安捷伦生物分析仪高灵敏度芯片对扩增后的cDNA文库进行质量评估与定量。之后将文库稀释至100 pM并进行等量混合，每个混合池包含4个独立样本；利用Ion Chef系统（Life Technologies）对混合文库完成扩增与富集；最后使用Ion PI HiQ试剂盒及3代芯片，在Ion Torrent Proton测序系统（Life Technologies）上完成模板化文库的测序。