Unknown,Transcriptomics,Genomics,Proteomics

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RNA sequencing comparing the effect of the introduction of RUNX1-D171N in Csf3r-d715 mice bone marrow cells

ABSTRACT: To address how Csf3r and RUNX1 mutations in combination with CSF3 administration affect hematopoiesis in vitro, we performed hematopoietic expansion cultures in LODISH stem cell expansion medium. Lineage-negative Csfr-d715 BM cell cells were retrovirally transduced with the patient specific RUNX1-D171N (RHD) mutant or an empty vector control and subsequently treated with puromycin to select for the transduced cells. Lineage- c-Kit+ (LK) cells were FACS sorted in TRIzol 2, 5 and 9 days after puromycin selection and subsequently used for RNA isolation according to the manufacturer’s protocol. SMARTer Ultra Low Input RNA kit for sequencing (Clontech, v4 Cat# 634891) was used to generate cDNA. Sequencing libraries were generated using TruSeq Nano DNA Sample Preparation kits (Illumina, Cat# 20015964), according to the low sample protocol and paired-end sequenced on a HiSeq 2500 (Illumina).

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Patricia Olofsen

PROVIDER: E-MTAB-9373 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO, and total RNA was isolated from a portion of the cells. The cells were further cultured in the presence of EPO, and RNA was isolated after cells differentiated into R3/R4 nucleated erythroid cells. There were 3 replicates of CD34 cells and 9 replicates of R3/R4 erythroid cells.

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RNA sequencing comparing the effect of the introduction of RUNX1-D171N in Csf3r-d715 mice bone marrow cells

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