Effect of SRSF2 mutations on splicing and gene expression in umbilical blood CD34+ cells

RNA-Seq was used to determine the effect of mutations in SRSF2 (P95H and P95R) that are associated with myeloid disease on the gene expression and alternative splicing. Umbilical cord CD34+ cells were transduced with lentiviral vectors expressing GFP, and GFP fusions of wild type SRSF2, SRSF2 P95H, or SRSF2 P95R. Seven days post transduction GFP positive cells were isolated by flow sorting. RNA was extracted from the sorted cells using the Total RNA Purification Plus Micro Kit (Norgen Biotek, Thorold, ON).rRNA depleted RNA-Seq libraries were generated using the Stranded RNA-Seq Kit with RiboErase (KAPA Biosystems, Wilmington, MA). Three replicate libraries, each derived from CD34+ cells obtained from a different donor, were generated for each set of samples. The libraries were subjected to 100 nt paired-end sequencing on Illumina Hi-Seq 4000.

本研究借助RNA测序（RNA-Seq）技术，解析与髓系疾病相关的SRSF2突变（P95H与P95R）对基因表达及可变剪接的调控效应。实验以脐带血CD34+细胞为研究材料，通过慢病毒载体分别转导表达绿色荧光蛋白（GFP）、野生型SRSF2、SRSF2 P95H或SRSF2 P95R的GFP融合蛋白。转导7天后，采用流式分选技术分离GFP阳性细胞。使用Total RNA Purification Plus Micro试剂盒（Norgen Biotek，加拿大安大略省索罗德市）从分选得到的细胞中提取总RNA。采用搭载RiboErase的链特异性RNA测序建库试剂盒（Stranded RNA-Seq Kit，KAPA Biosystems，美国马萨诸塞州威尔明顿市）构建核糖体RNA（rRNA）去除型RNA测序文库。每组样本对应3个生物学重复文库，每个文库的细胞均来源于不同供者的CD34+细胞。将构建完成的文库置于Illumina HiSeq 4000测序平台，进行100 nt双端测序。