Project description:To study the effects of miR--210 on 293T cells and hypoxia response, we used the CRISPR/Cas9 system to knockout (KO) the human miR-210 gene in 293T cells. We then added DMOG to mimic hypoxia condition and analyzed the RNA expression profile of wildtype (WT) and KO cells under normal and hypoxic conditions. DMOG treatment and RNA-seq were performed three times (experiment 1, 2, and 3).
Project description:293T cells are transfected with hsa_circ_0001400 expression plasmid vectors for 24 hours. Cells are then incuvated with Psoralen, exposed to long-wave UV for 10 minutes, and total RNA was extracted. Biotynilated DNA oligos sense (control) or antisense of circ_0001400 are added and circRNA-RNA complexes are enriched using streptavidin beads. RNA was cleaned and exposed to short-wave UV and subjected for RNA-Seq.
Project description:The mechanism of mecciRNA degradation remains unknown. To investigate the degradation of mecciRNAs, we performed mitochondrial RNA sequencing and total RNA sequencing of 293T, HeLa, and N2a cells. To investigate the degradation mechanism of mecciRNAs, RIP-seq was conducted in 293T, HL-1 cells, and C. elegans. Small RNA sequencing of mitochondrial sucrose gradient fractions was performed to identify mecciRNA degradation fragments.
Project description:MicroRNAs (miRNAs) bind to the 3'-untranslated region of target mRNAs in a sequence-specific manner and subsequently repress gene translation. Human miR-26a has been studied extensively, but the target transcripts are far from complete. We first employed the CRISPR-Cas9 system to generate an miR-26a-knockout line in human cervical cancer HeLa cells. The miR26a-knockout line showed increased cell growth and altered proliferation. Proteomics technology of sequential window acquisition of all theoretical mass spectra (SWATH-MS) was utilized to compare the protein abundance between the wild-type and the knockout lines, with an attempt to identify transcripts whose translation was influenced by miR-26a. Functional classification of the proteins with significant changes revealed their function in stress response, proliferation, localization, development, signaling, etc. Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring (PRM). The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification. Among the computationally predicted targets, a subset of the targets was directly regulated by miR-26a, as demonstrated by luciferase assays and Western blotting. This study creates an inventory of miR-26a-targeted transcripts in HeLa cells and provides fundamental knowledge to further explore the functions of miR-26a in human cancer.
Project description:Gene Expression changes in BMDCs stimulated with H. pylori vs. E. coli. The hypothesis tested was that the gene expression profile in BMDCs stimulated with H. pylori lysate will be less inflammatory than BMDCs stimulated with E. coli. BMDCs were generated from bone marrow of wild type female C57BL/6 mice using 100ng/mL Flt3L. DCs were harvested at day 8 and stimulated with either media alone, H. pylori antigen lysate or E. coli lysate. At 24 hours, the cells were harvested and RNA was isolated for microarray analysis.
Project description:We present four datasets on proteomics profiling of HeLa and SiHa cell lines associated with the research described in the paper "PROTREC: A probability-based approach for recovering missing proteins based on biological networks" [1]. Proteins in each cell line were acquired by two different data acquisition methods. The first was Data Dependent Acquisition-Parallel Accumulation Serial Fragmentation (DDA-PASEF) and the second was Parallel Accumulation-Serial Fragmentation combined with data-independent acquisition (diaPASEF) [2], [3]. Protein assembly was performed following search against the Swiss-Prot Human database using Peaks Studio for DDA datasets and Spectronaut for DIA datasets. The assembled result contains identified PSMs, peptides and proteins that are above threshold for each HeLa and SiHa sample. Coverage-wise, for DDA-PASEF, approximately 6,090 and 7,298 proteins were quantified for HeLa and SiHA sample, while13,339 and 8,773 proteins were quantified by diaPASEF for HeLa for SiHa sample, respectively. Consistency-wise, diaPASEF has fewer missing values (∼ 2%) compared to its DDA counterparts (∼5-7%). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository [4] with the dataset identifier PXD029773.
Project description:Limited proteolysis coupled to mass spectrometry (LiP-MS) is a structural proteomics technique that can be used to identify the targets of small molecules. This is achieved by incubating lysate with the drug of interest, which induces structural changes such as occupation of the binding site or changes and protein-protein interactions. This is followed by a limited proteolysis step in which the unspecific protease proteinase K is added to the treated lysate. The protease preferentially cleaves accessible and flexible regions of the proteins, thus generating peptides that reflect the changes in surface accessibility caused by the treatment with the small molecule. Here we treated HEK293 cell lysate with rapamycin and identify the known main target FKBP1A. This data is intended to be used as a model dataset for LiP-MS data evaluation and was created as part of the IMI EUbOPEN project.
Project description:Transcriptional profiling of Human Embryonic Kidney(293T) Cells comparing control untreated 293T cells with 293T cells transfected with A) pcDNA 3.0(-) vector[Invitrogen] (Mock) and B) Expression vector pcDNA 3.0(-) containing cloned Influenza virus H5N1and H11N1-NS1 (Non-Structural1) gene.