Project description:3 µl of each human serum sample was added to 40 µl lysis buffer containing 5% sodium dodecyl sulfate (SDS) and 50 mM triethylammonium bicarbonate (TEAB), pH 8.5. Proteins were reduced by addition of 15 mM dithiothreitol and incubation for 30 minutes at 55˚C and then alkylated by addition of 30 mM iodoacetamide and incubation for 15 minutes at RT in the dark. Phosphoric acid was added to a final concentration of 1.2% and subsequently samples were diluted 7-fold with binding buffer containing 90% methanol in 100 mM TEAB, pH 7.55. The samples were loaded on the 96-well S-Trap™ plate (Protifi), placed on top of a deepwell plate, and centrifuged for 2 min at 1,500 x g at RT. After protein binding, the S-trap™ plate was washed three times by adding 200 µl binding buffer and centrifugation for 2 min at 1,500 x g at room temperature. A new deepwell receiver plate was placed below the 96-well S-Trap™ plate and 50 mM TEAB containing trypsin (1/100, w/w) was added for digestion overnight at 37°C. Using centrifugation for 2 min at 1,500 x g, peptides were eluted in three times, first with 80 µl 50 mM TEAB, then with 80 µl 0.2% formic acid (FA) in water and finally with 80 µl 0.2% FA in water/acetonitrile (ACN) (50/50, v/v). Eluted peptides were dried completely by vacuum centrifugation. Dried peptides were redissolved in 100 µl 0.1% FA, the peptide concentration was determined on a Lunatic spectrophotometer (Unchained Labs) and was adjusted to 0.015 µg/µl with 0.1% FA1. iRT peptides (Biognosys, P/N Ki-3002-1) were added to all samples according to the manufacturer’s instructions. Then, 300 ng of each sample was loaded on Evotips (Evosep, P/N EV2003) according to the manufacturer’s instructions with a substitution of the wash step after sample loading by two wash steps with 80 µl 0.1% FA. All loaded Evotips were stored in 0.1% FA at 4°C until LC-MS/MS analysis was started.
Project description:To study their metabolic potential in natural ecosystems, we developed a species-independent LAB microarray, containing 2,269 30-mer oligonucleotides, and targeting 406 genes that play a key role in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in stress response. Also, genes linked to negative traits such as antibiotic resistance and virulence are represented. This experiment is a validation experiment, where we hybridized labelled RNA from 20 LAB strains, covering 86% of all oligos. Keywords: Platform validation experiment
Project description:To study their metabolic potential in natural ecosystems, we developed a species-independent LAB microarray, containing 2,269 30-mer oligonucleotides, and targeting 406 genes that play a key role in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in stress response. Also, genes linked to negative traits such as antibiotic resistance and virulence are represented. This experiment is a validation experiment, where we hybridized labelled DNA from 20 LAB strains, covering 86% of all oligos. Keywords: Platform validation experiment
Project description:Formaldehyde (FA) is a commercially important chemical with numerous and diverse uses. In this study, a functional toxicogenomics approach was utilized in the model eukaryotic yeast Saccharomyces cerevisiae to identify genes and cellular processes modulating the cellular toxicity of FA. Our results demonstrate mutant strains deficient in multiple DNA repair pathways were sensitive to FA. The SKI complex and its associated factors, which regulate mRNA degradation by the exosome, were also required for FA tolerance..
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Jonathan Preall jpreall@cshl.edu (Generation 0 Data from Hannon Lab), Carrie Davis davisc@cshl.edu (experimental), Alex Dobin dobin@cshl.edu (computational), Wei Lin wlin@cshl.edu (computational), Tom Gingeras gingeras@cshl.edu (primary investigator)). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). hg18: This data was produced by Hannon lab part of Cold Spring Harbor as part of the ENCODE Project. The series depicts NextGen sequencing information for RNAs between the sizes of 20-200 nt isolated from RNA samples from tissues or sub cellular compartments of cell lines. hg19: This track depicts NextGen sequencing information for RNAs between the sizes of 20-200 nt isolated from RNA samples from tissues or sub cellular compartments from ENCODE cell lines. The overall goal of the ENCODE project is to identify and characterize all functional elements in the sequence of the human genome. hg19: This cloning protocol generates directional libraries that are read from the 5' ends of the inserts, which should largely correspond to the 5' ends of the mature RNAs. The libraries were sequenced on a Solexa platform for a total of 36, 50 or 76 cycles however the reads undergo post-processing resulting in trimming of their 3' ends. Consequently, the mapped read lengths are variable. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf