A comparison of the effects of duplex-specific nuclease, antisense oligonucleotides, and riboZero rRNA depletion on ribo-Seq data from mouse
ABSTRACT: Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. A key step is the depletion of ribosomal RNA that allows sufficient depth of sequencing of the mRNA undergoing ribosomal translation. These data are designed to compare four strategies for ribosomal depletion; a novel strategy based on duplex-specific nuclease using either one or two cycles, the antisense oligonucleotides described in Ingola et al (2012) and the commercially available RiboZero kit (with a no treatment control).
Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, ...[more]
Project description:Ribosome profiling (RiboSeq) maps positions of translating ribosomes on the transcriptome. Here we optimized ribosome profiling for footprinting mitochondrial ribosomes, and profiled three human cell-lines - HEK293, a PDE12-/- knockout, and a deltaFLP 143B cybrid.
Project description:The WhiG sigma factor gene is required for spore formation is Streptomyces venezuelae. It is similar to the FliA sigma factor of E. coli. WhiG deletion strains are able to make aerial hyphae but are defective in the spore maturation. This ChIP-Seq experiment was carried out to determine all the binding sites WhiG binds to in the genome of Streptomyces venezuelae. Anti-WhiG polyclonal antibodies were used for ChIP-Seq of the wild type (WT) strain after 34 hours of growth in shaken cultures. A WhiG deletion strain was made and anti-WhiG antibodies were used for ChIP-Seq in the deletion strain after 34 hours of growth in shaken cultures. This was used as the negative control and ChIP-Seq peak positions in this were disregarded in the WT.
Project description:The BldC gene is required for the formation of aerial hyphae Streptomyces venezuelae. It is 68 amino acid DNA-binding protein related to the MerR family of transcription factors. BldC deletion strains are bald because they initiate sporulation prematurely, omitting the formation of aerial hyphae altogether. This ChIP-Seq experiment was carried out to determine all the binding sites BldC binds to in the genome of Streptomyces venezuelae. Anti-BldC antibodies were used for ChIP-Seq of the wild type (WT) strain after 10 and 14 hours of growth in shaken cultures. A BldC deletion strain was made and anti-BldC antibodies were used for ChIP-Seq in the deletion strain after 14 hours of growth in shaken cultures. This was used as the negative control and enrichment in this ChIP-Seq was subtracted from the enrichment in the WT strain.
Project description:Genome-wide identification of RNA polymerase (RNAP) binding sites were performed in Klebsiella pneumoniae MGH 78578 (KP). Anti-RNAP is used to capture the RNAP in KP. ChIP-chip was performed on tiling array specifically made for KP. Comparison ChIP by anti-RNAP antibody vs ChIP by normal mouse IgG (control, mock IP)
Project description:Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here, we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but employs duplex-specific nuclease as a convenient, bias-free, species-independent way to reduce rRNA contamination. Our protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream and overlapping open reading frames (ORFs). This feature also allows the identification of an alternative ribosome conformation evident during termination of protein synthesis. To test the effectiveness of DSN in ribosome profiling, we generated Ribo-Seq libraries from mouse tissue culture cells and from the green alga Chlamydomonas reinhardtii.
Project description:Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. In contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, we found that oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103+CD11blow (CD103+LCs) and CD11b+CD103- (CD11b+LCs) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103+LCs originate from pre-DCs, whereas CD11b+LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with their epithelial position, morphology and expression of LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DCs and monocytic) in steady state The following cells were isolated from mice (2-4 replicates): Lung DCs, mucosal CD103+ LC, mucosal CD11b+ LC, Skin LC. Transcriptome analysis was performed.
Project description:P388D1 murine macrophages were cultured in 85 mm tissue culture plates to about semi confluency. L. monocytogenes serotypes (1/2a EGD-e, 4a L99, 4b CLIP80459 and 4b F2365) were infected to the P388D1 cell monolayer at a MOI of 100 per eukaryotic cell. Infection was carried for 45 min and followed by addition of fresh medium containing 20 µg/ml gentamicin. The medium of the plates (containing 20 µg/ml gentamicin) infected with L. monocytogenes serotypes were replaced after 2 h post infection with fresh medium containing 50 µg/ml gentamicin. At each step the plates were washed extensively with 1x PBS. Incubation of the bacterial tissue culture plates was carried out in a humidified incubator for up to 4 h post infection.
Project description:Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. Part of the explanation probably lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicated that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses revealed that SF3B1 is specifically bound to nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affected the splicing of these exons similarly to inhibition of SF3B1 expression. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure. MNase-seq on Input and SF3B1 pull-down, mRNA-seq on control and SF3B1 si-RNA treated cells as well as on TSA (Trichostatin A) treated and untreated cells.