Project description:How Krüppel-associated box-domain zinc finger protein (KRAB-ZFP) transcriptional repressors recruit TRIM28/KAP1 to recognizes target sequences or foreign genomes is unclear. Here, we used Epstein-Barr virus-infected cells in which a KRAB-ZFP, SZF1, silences lytic/replicative-phase genes of the virus to determine how the ZFP-footprints on cell and viral genomes to target pericentromeric regions. We observed that it uses non-consensus sites to target viral genes. This heterochromatin machinery employed by the host silences and regulate an invader without deregulating itself.
Project description:In order to identify SOX4- and TGF-beta dependent genes, human mammary epithelial cells (HMLEs) were transduced with shRNA scrambled (control) or shRNA targeting SOX4 (TRCN0000018213, Sigma Aldrich, St. Louis, MS) and selected with puromycin for at least 2 weeks. SOX4 knockdown was confirmed by qRTPCR and western blotting. Cells were plated in 6 well plates and either left untreated or treated with TGF-beta (2.5ng/ml) for 16 hours in biological duplicate (different passages). Samples were harvested and total RNA was isolated using RNAeasy kit (Qiagen). Samples were sequenced on the NextSeq 500 (Illumina), SE75.
Project description:U2-OS cells were transfected with scramble siRNA or PKC delta siRNA, then left untreated or treated with 2 ug/ml adriamycin for 8 h.
Project description:Jurkat T cells (in triplicate per treatment group) were left untreated in culture, infected with VSV-G-pseudotyped HIV-based vector (which transduces Tat and eGFP) or treated with TNFalpha. Keywords: parallel sample
Project description:The emerging evidences support that exosome cargo miRNAs function as important regulators in cell differentiation. Therefore, in order to figure out the mechanism that Exo-AT mediated adipogenesis, we profiled miRNAs in Exo-AT using high-throughput sequencing (miRNA-seq). After trimming low-quality reads, contaminants, adaptors, and reads smaller than 15 nt, the remaining reads were mapped to merged pre-miRNA data bases. To identify the conserved miRNAs in Exo exosomes, miRNAs were aligned to miRBase v21. 148 and 154 types of known miRNAs in Exo-ADSCs and Exo-AT, respectively, were identified in the two replicates. Among these miRNAs, 103 miRNAs were simultaneously detected in both Exo-ADSCs and Exo-AT. Compared to Exo-ADSCs, 45 conserved miRNAs were enriched (expressed ≥ 2 folds, FDR<0.05) in Exo-AT. KEGG Pathway analysis was performed for the targets of the most 20 enriched miRNAs in Exo-AT (compared with Exo-ADSCs) to determine their potential function. Data showed that pathways that regulate adipogenesis such as Wnt signaling pathway, Insulin signaling pathway, MAPK signaling pathway, TGF-ß signaling pathway were enriched significantly for targets of Exo-AT miRNAs. Furthermore, 14 of 45 enriched miRNAs in Exo-AT (31.11%, such as miR-30a-5p, miR-148a-3p) were reported to participate in regulation of adipogenesis while 8 miRNAs (17.78%, such as miR-93-5p, miR-150-3p) that negatively control osteoblastic differentiation of MSC have been described.
Project description:Wild-type C57BL/6N mice were treated with twice-weekly carfilzomib injections for a period of two weeks. Following this treatment, the left ventricular tissues were prepared for tmt-multiplexed mass spectrometry analysis to determine the protein-profiles of the treated hearts as opposed to those of untreated mouse specimens. For more experimental details and procedures, please see the attached methods file.
Project description:To explore the possibility of miRNA(s) contributing to the cardioprotection induced by plasma exosomes at the late phase of RIPC, we performed a miRNA profiling assay (763 rat miRNAs) comparing the differences between RIPC-exo and Control-exo using Illumina HiSeq 2500 high-throughput sequencing.
Project description:Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.