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.