Project description:Severe congenital neutropenia (CN) is a pre-leukemia syndrome that, in the majority of patients, is caused by heterogeneous ELANE mutations encoding neutrophil elastase (NE). To study leukemogenesis associated with CN we generated CN and CN/AML patient-specific induced pluripotent stem cells (iPSCs). Additional mutations in leukemia-relevant genes, CSF3R and RUNX1, were introduced using CRISPR/Cas9 gene-editing. Consequently, we performed in vitro embryoid body (EB)-based hematopoietic and myeloid differentiation of generated iPSC lines. On day 14-17 of EB-based differentiation, iPSC-derived CD45+CD34+ cells were harvested and mRNA was isolated using RNeasy Mini- or Micro Kit (Qiagen). Sequencing libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina). Poly (A) selected single-read and pair-read sequencing libraries were sequenced on the Illumina platform in order to compare the transcriptomes of CN and CN/AML iPSCs-derived HSPCs from 2 CN/AML patients. Next, we identified that BAALC knockout resulted in a dramatic induction of granulocytic differentiation and a significant reduction in proliferation of CN/AML iPSC-derived HSPCs. To identify BAALC-dependent leukemia-associated gene expression, we compared the transcriptomes of CN/AML iPSCs before and after BAALC KO using a similar approach described above for CN and CN/AML iPSCs-derived HSPCs.
Project description:To identify genes involved in nutrition metabolism of Macrobrachium nipponense, two independent cDNA libraries from hepatopancreas and muscle were constructed through high-throughput next-generation sequencing techniques. A total of 112,548,142 and 90,140,774 high quality reads were generated in the two cDNA libraries, respectively. Clustering and assembly of these reads produced a nonredundant set of 98,560 unique sequences, with an average unigene length of 793 bp. The unigene sequences were subjected to GO, COG and KEGG functional classification. A large number of differentially expressed genes were recovered by comparison of the two tissues. The differentially expressed and their functions were predicted by KEGG pathway mapping. Furthermore, 17,971SSRs and 32,935 high-confidence SNPs were identified in the two EST datasets. This study lays the foundation for further research on gene function analysis in nutrition metabolism of M. nipponense. sample 1: hepatopancreas; sample 2: muscle
