Project description:Inactivating EZH2 mutations have been associated with myelofibrosis (MF). Moreover, EZH2 mutations co-exist with the JAK2V617F mutation in a significant cases of MF. To determine the effects of concomitant loss of EZH2 and JAK2V617F mutation in hematopoiesis, we generated Ezh2-deficient Jak2V617F-expressing mice. To identify the Ezh2 target genes that are regulated by H3K27me3 in MF, we performed H3K27me3 ChIP-sequencing in sorted LSK cells from control, MxCre;Jak2VF/+ and MxCre;Jak2VF/+ EZH2-/- mice. Overall design: 1500μg pI-pC was administrated to control, MxCre Jak2VF/+ and MxCre Jak2VF/+ EZH2-/- at 4 weeks after birth in five times (300μg each time). Bone marrow cells were harvested from the mice at 12 weeks after pI-pC injections.ChIP-seq experiments were performed for LSK cells sorted from BM cells.
Project description:Inactivating EZH2 mutations have been associated with myelofibrosis (MF). Moreover, EZH2 mutations co-exist with the JAK2V617F mutation in a significant cases of MF. To determine the effects of concomitant loss of EZH2 and JAK2V617F mutation in hematopoiesis, we generated Ezh2-deficient Jak2V617F-expressing mice. To gain insights into the mechanisms by which Ezh2 deficiency promotes the development of MF in Jak2V617F knock-in mice, we performed microarray gene expression analysis on sorted LT-HSC from control, MxCre;Jak2VF/+ and MxCre;Jak2VF/+ EZH2-/- mice. Overall design: Microarray analyses were performed on LT-HSC sorted from control, MxCre Jak2VF/+ and MxCre Jak2VF/+ EZH2-/- mice BM cells at 12 weeks after pI-pC injections. Three biological replicates for each genotype, i.e., a total of nine samples were utilized for microarray analyses.
Project description:The JAK2V617F mutation has been detected in ~50% cases of MF. Elevated expression of high mobility group AT hook 2 (HMGA2) also has been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2V617F knock-in mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. In order to assess the effects of Hmga2 overexpression on gene expression, we performed RNA sequencing analysis for the LSK from Jak2VF/+ vector and Jak2VF/+ Hmga2mice. Overall design: Bone marrow cells from 5-fluorouracil-primed Mx1Cre; Jak2V617F/+ (referred as Jak2VF/+) mice were transduced with lentiviruses expressing vector alone or Hmga2 by two rounds of spin infection. Transduced bone marrow cells (1X106) were injected into retro-orbital veins of lethally irradiated (2X 550 cGy) C57BL/6 recipient mice. BM cells were harvested at 32 weeks after BMT. RNA sequencing analysis from Jak2VF/+ vector and Jak2VF/+ Hmga2 mice were performed for LSK cells sorted from BM cells. There are three biological replicates for each genotype and six samples in total.
Project description:Identifying putative transcription factor target genes by combining CRISPR/Cas9-based transcriptional activation with RNAseq in Drosophila S2R+ cells. This study focuses on the transcription factors Twist and Snail, singly and together. RNA from Drosophila cells following CRISPR/Cas9-based activation of Twist, Snail, or Twist and Snail together, compared with non-targeting sgRNA. Two biological replicates for each experiment
Project description:We sequenced polyA mRNA from OVCAR8-ADR-Cas9 cells in which one or two of 3 epigenetic regulators (BRD4, KDM4C, KDM6B) had been knocked out to examine how global gene expression was affected and evaluate potential synergistic effects at a molecular level. Gene expression data (RNA-Seq) in OVCAR8-ADR-Cas9 cells infected with control vector or vectors expressing gRNAs targeting one of 4 epigenetic regulators (BRD4, KDM4C, KDM6B) with biological replicates.
Project description:RNA-Seq after Cas9-gRNA transfection with different length gRNAs we performed PolyA Selection and RNA-Seq on cells transfected with dCas9-VPR and a gRNA of each length (20nt, 16nt, or 14nt) targeting ACTC1, MIAT, or HBG1/2