Project description:Purpose: A method for mapping chromatin accessibility genome-wide, to reveal chromatin accessibility in Intestinal stem cells. Methods: Intestinal stem cells(Lgr5-high cells) were sorted by flow cytometry from wild type mice. The samples were prepared in duplicate. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage was used to generate bigwig files from bam files. MACS2 (v2.2.5) was used for peak calling and to generate bed files from aligned reads. Conclusions: ATAC-seq analysis confirmed that Fosb binding sites in Chip-seq assay were correlated with the chromatin accessibility .
Project description:Reactive oxygen species (ROS) are involved in the regulation of diverse physiological processes in plants, including various biotic and abiotic stress responses. Thus, oxidative stress tolerance mechanisms in plants are complex, and diverse responses at multiple levels need to be characterized in order to understand them. The MS raw data files were converted to mzXML files using massWolf (version 4.3.1). The MS mzXML and MASCOT xml files were parsed and processed with a program developed in-house. Briefly, each scan was subjected to smoothing using Savitzky-Golay filtering (second order polynomial, five data points, two iterations) and peak areas were calculated after noise reduction. Peak mass was set to the average of the three highest data points for each peak. Unique peptides identified with MASCOT were matched to the parsed MS data using the parameters detected m/z, charge state and retention time, using a retention time window of ± 1.0 min. Charge states were calculated by using the first three isotopic peaks of a peptide and the same mass tolerances for detecting the mono isotopic peak as in the MASCOT search.
Project description:Purpose: A method for identifying genome-wide DNA binding sites for Fosb. Methods: Alive cells were sorted from retro-Fosb-OE(over-expression) organoids. The samples were incubated with anti-Fosb antibody (Abcam, ab184938). Purified DNA was subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep Kit and sequenced as paired-end with Illumina Novaseq 6000. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage (CPM normalized and extended reads) was used to generate bigwig files from bam files. MACS2 was used for peak calling and to generate bed files from aligned reads. HOMER annotatePeaks.pl was used to annotate the peaks. Conclusions: Target genes of Fosb through ChIP assay were consistent with predicted target genes. Thus, we concluded that Fosb, which is a key TF could regulate most of ISC signature genes to maintain Lgr5+ ISCs.
Project description:Legume seeds and peanuts, in particular, are an inexpensive source of plant proteins and edible oil. Owing to their importance in global food security, it is necessary to understand the genetic, biochemical, and physiological mechanisms involved in controlling seed quality and nutritive attributes. A comprehensive understanding of seed development and the effects of water-deficit stress on the incorporation of the main storage reserves in seeds, such as proteins, fatty acids, starch, and secondary metabolites will enhance our ability to improve seed quality and yield through molecular breeding programs. In the present study we employed a label-free quantitative proteomics approach to study the functional proteins altered in the mid mature peanut seed during water-deficit stress. The RAW files of LC-MS/MS runs were converted to mzXML format and searched using GPM (Global Proteome Machine, version 2.1.1) software. Each mzXML spectra file was also searched against a reversed sequence database to calculate the false discovery rate (FDR). The 16 output files for each replicate were combined to create a single merged result file and only proteins with FDR less than 1% were used for the analysis. The following parameters were used for the search: Enzyme: trypain, [RK]|{P}; allowed missed cleavage: 2; variable modification: methionine oxidation; fixed modification: carbamidomethylation of cysteine; MS and MS/MS mass tolerance: ±2 Da and ±0.2 Da respectively. For the comparative analysis, the normalized spectral abundance factors (NSAFs) was used to study protein abundance as described earlier. For each identified protein, k, in sample i, the number of spectral counts was divided by length of the identified protein. NSAFi values for each sample i were obtained by normalizing SpCk/Lengthk values to the total by dividing by the sum (SpCk/Lengthk) over all proteins.
Project description:Kinases are a major clinical target for human diseases. Identifying the proteins that interact with kinases in vivo will provide information on unreported substrates and will potentially lead to more specific methods for therapeutic kinase regulation. Here, endogenous immunoprecipitations of evolutionally distinct kinases (i.e. Akt, ERK2, and CAMK2) from rodent hippocampi were analyzed by mass spectrometry to generate three highly confident kinase protein-protein interaction networks. Proteins of similar function were identified in the networks, suggesting a universal model for kinase signaling complexes. Structural connections were observed between kinases with reported symbiotic relationships. The kinase networks were significantly enriched in genes associated with specific neurodevelopmental disorders providing novel structural connections between these disease-associated genes. To demonstrate a functional relationship between the kinases and its network, pharmacological manipulation of Akt in hippocampal slices was shown to regulates the activity of potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel(HCN1), which was identified in the Akt network. Overall, the kinase protein-protein interaction networks provide molecular insight of the spatial complexity of in vivo kinase signal transduction which is required to achieve the therapeutic potential of kinase manipulation in the brain.
Project description:We profile transcriptome, proteome and phosphoproteome in a panel of non-small cell lung cancer (NSCLC) cell lines in order to reconstruct targetable networks associated with KRAS dependency. We develop a two-step bioinformatics strategy addressing the challenge of integrating these disparate datasets. We first define an “abundance-score” combining transcript, protein and phospho-protein abundances to nominate differentially abundant proteins and then use the Prize Collecting Steiner Tree algorithm to identify functional sub-networks. We identify three modules centered on KRAS and MET, LCK and PAK1 and Beta-catenin. We validate activation of these proteins in KRAS-dependent cells and perform functional studies defining LCK as a critical gene for cell proliferation in KRAS-dependent but not KRAS-independent NSCLCs. These results are the first evidence that suggest LCK as a potential druggable target protein in KRAS-dependent lung cancers. Data analysis: Raw spectra files were converted to mzXML using ReadAW. The mzXML files were searched using X!Tandem with the k-score plug-in. The proteomic searches were performed using the following options: allow up to 2 missed tryptic cleavages, a parent ion tolerance window of -1 to +4 Daltons, and a fragment ion tolerance of 0.8 Da. The following variable modifications were allowed: phosphorylation of Serine, Threonine, and Tyrosine (+79.966331@[STY]), oxidation of Methionine (+15.994920@M), and carbamidomethylation of Cysteine (+57.021460@C). All protein searches were performed using the Human Refseq protein database (release 47). Appended to this database were common proteomic contaminants and reversed protein sequences to serve as decoys. The X!Tandem results were then post processed with PeptideProphet and ProteinProphet.
Project description:Data was acquired using Q Exactive and C18 RP-UHPLC in positive mode.
mzXML files only.
Data used a test set for Ion Identity Molecular Networking.
Project description:Temporal analysis of Irf4 and PU.1 genome binding during B cell activation and differentiation in vitro using antigen (NP-Ficoll) CD40L and IL-2/4/5 cytokines (see Molecular Systems Biology 7:495 for details of cellular system). The results provide insight in the target genes and binding specificity of IRF4 and PU.1 during coordination of different programs of B cell differentiation. Regrettably three of the FASTQ raw sequence files in our study were corrupted during storage. FASTQ data from our experimental and control groups are available for download via GEO SRA; however, two groups are missing select raw sequence files. These include one PU.1 Day 3 group file (Sample GSM1133499) and two of four input files used to generate a concatenated “super” input file (Sample GSM1133490); the raw data provided for input consists of the two input files recovered. Importantly, FASTA sequences for both of these datasets are available as supplementary data through GEO, and we can make available upon request (rsciamma@uchicago.edu) all files in our study in the ELAND-extended alignment format. Please note that GEO no longer supports this format.