Project description:H157 cells transfected with vector and overexpressed AACT plasmids were treated and used for MS detection. The MS data were acquired using an Orbitrap Exploris 480 Mass Spectrometer coupled with an EASY-NLC 1200 liquid phase LC-MS system. DIA raw data files were analyzed by DIA-NN software. The differentially expressed proteins were screened in this quantitative proteomics.
Project description:To identify direct transcriptional targets of RFX6, we performed chromatin immunoprecipitation of HA epitope tagged RFX6 followed by massively parallel DNA sequencing (ChIP-seq). Using CRISPR/Cas9 gene editing, the HA epitope was inserted into the 3' end of the RFX6 gene in H9 hESC. Pluripotent cells were then differentiated into PDX1+RFX6+ pancreatic progenitors and endogenous RFX6-HA was immunoprecipitated with an anti-HA antibody. To eliminate background signal caused by non-specific antibody binding, a control experiment using wild-type H9 hESC was performed in parallel.
Project description:To identify those proteins interacting with the cytoplasmic dynein-2 using mammalian cell lines stably expressing HA-tagged intermediate chain subunits, WDR34 (DYNC2I2) or WDR60 (DYNC2I1).
Project description:We used miRNA microarrays to detail the miRNA content in A549 Ago2-KO/HA-Ago2Wt, Ago2-KO/HA-Ago2KA, and Ago2-KO/HA-Ago2Δ (Dm) cells
Project description:Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers and epigenetic marks. To measure dissociation rates of H3.3, we utilized a TET-repressible ESC line, ES[MC1R(20)], with the expression cassette integrated at the ROSA26 locus. We transfected MC1R ESCs with HA/FLAG-tagged H3.3 controlled by tetracycline response elements. For ‘TET-OFF’ experiments, ESCs were cultured over several passages (weeks) on feeder cells in the absence of DOX and were subsequently passaged onto feeder-free plates prior to the inhibition of HA-H3.3 expression. To repress HA/FLAG-H3.3 expression we treated cells with 2 ?g/ml doxycycline hyclate before crosslinking with formaldehyde at various time points. Measurement of H3.3-HA enrichment over time-course was performed using two replicates of ChIP-seq experiments. As a control, input DNA sequencing was performed for every time point
Project description:TFE3 is a bHLH-ZIP transcription factor, which nuclear localization is regulated by a tumor suppressor FLCN. In order to analyze TFE3 occupancy in whole genome, we have generated and utilized a HK-2 HA-TFE3-inducible cell line which express HA-tagged TFE3 in a doxycycline-dependent manner. HA-TFE3 bound regions were determined by ChIPSeq.