Project description:Grad-seq in Clostridium difficile 630. Cell lysate is analyzed in a gradient and fractionated into 21 fractions which are analysed for proteins by MS and for transcripts by RNA-sequencing.
Project description:Proteomic data of ScSUGAR-seq samples: 1)TMT experiment to confirm changes in glycosylation due to Swainsonine 2)LFQ biotin pull down experiment to assess lectin binding partners 3)LFQ of MGAT1 cell lines assessing glycopeptide and glycoproteome level changes
Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Phages are important drivers of bacterial evolution with therapeutic potential as antimicrobials. However, gaps in our understanding of phages and ability to rapidly engineer them with new genetic cargo hinders progress towards phage-based therapies. To address the lack of unbiased, genome-wide mutational tools for phages, we developed transposon mutagenesis employing CRISPR-anti-CRISPR (Acr)-based selection and deep-sequencing (Phage Tn-seq). Transposon mutagenesis was effective for phages with unmodified or hypermodified genomes and a jumbo phage that protects its DNA within a nucleus. Phage Tn-seq enabled phage gene essentiality assignment consistent with structural proteomics and core gene conservation. Insertion biases allowed prediction of transcriptional direction and early injected phage DNA regions. We exploited the method to rapidly deliver new cargo to phage genomes in just a few days and used an AI-designed Acr to expand the phage transposon toolbox. Phage Tn-seq is versatile tool to advance our understanding and applications of phages.
Project description:Here we analyzed the full ensemble of cellular RNAs and co-fractionating proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803, a cyanobacterium rich in internal membrane systems.
Project description:Energy metabolism and extracellular matrix function are closely connected to orchestrate and maintain tissue organization, but the crosstalk is poorly understood. Here, we used scRNA-seq analysis to uncover the importance of respiration for extracellular matrix homeostasis in mature cartilage. Genetic inhibition of respiration in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocyte clusters. These changes were associated with alterations in extracellular matrix composition, a shift in the collagen/non-collagen protein content and an increase of collagen crosslinking and ECM stiffness. The results demonstrate, based on findings of the scRNA-seq analysis, that respiration is a key factor contributing to ECM integrity and mechanostability in cartilage and presumably also in many other tissues.