Project description:Circadian clocks are important for gut health. This experiment aimed to determine the role of core clock gene Bmal1 in regulating microbial rhythmicity in health and dextran sulphate sodium induced colitis. Mice were generated with Bmal1 selectively deleted in Villin-expressing cells (predominantly IECs).Microbial DNA was extracted from fecal pellets collected from IEC-Bmal1-/- and Bmal1flox mice (aged 8-19 weeks) at zeitgeber time 0, 4, 8, 12, 16, 20 across the 24h day with the DNeasy PowerSoil Pro Kit (Qiagen), as per manufacturer’s instructions. Pre-amplification of the V4 region of 16S rRNA was performed using forward primer 5'-ACACTCTTTCCCTACACGACGCTCTTCCGAT-CTNNNNNGTGCCAGCMGCCGCGGTAA-3' (annealing sites in bold) and reverse primer 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT-3'. Sequencing was performed by the University of Liverpool Centre for genomics Research, using the Illumina MiSeq v2 platform (Illumina), generating 250bp paired-end reads. PhiX control v3 library (PhiX) was spiked into samples to balance low base diversity often found in microbiome samples. Quality control was performed and OTU tables were generated using a pipeline provided by the University of Manchester Bioinformatics Core Facility local Galaxy service. Briefly, VSEARCH clustered OTUs and removed chimeras. The OTU database was mapped to the SILVA (v138) reference database with >97% homology threshold. All samples passed quality checks and had sequence depth >45,000. The OTU table was analysed using R packages phyloseq, vegan, limma and ALDEx2. JTK_CYCLE 93 was used to identify rhythmic OTUs with a period of 24 h and an adjusted P value < 0.05.
Project description:This study investigates the gut microbiome composition and diversity in three groups of rats: control, radiation enteritis model, and treatment (TG) groups. Total DNA was extracted from stool samples, PCR-amplified targeting 16S rRNA gene variable regions, and sequenced using Illumina MiSeq or NovaSeq platforms. Downstream bioinformatics analyses included sequence quality control, denoising (DADA2/OTU clustering), taxonomic classification, alpha and beta diversity evaluation, differential species abundance analysis, and functional prediction. The processed data include ASV/OTU tables, taxonomy assignments, and sample metadata.
Project description:The rise of sample multiplexing in quantitative proteomics for the dissection of complex phenotypic comparisons has been advanced by the development of ever more sensitive and robust instrumentation. Here, we evaluated the utility of the Orbitrap Eclipse Tribrid mass spectrometer (advanced quadrupole filter, optimized FTMS scan overhead) and new instrument control software features (Precursor Fit filtering, TurboTMT and Real-time Peptide Search filtering). Multidimensional comparisons of these novel features increased total peptide identifications by 20% for SPS-MS3 methods and 14% for HRMS2 methods. Importantly Real-time Peptide Search filtering enabled a ~2X throughput improvement for quantification. Across the board, these sensitivity increases were attained without sacrificing quantitative accuracy. New hardware and software features enable more efficient characterization in pursuit of comparative whole proteome insights.
Project description:Preeclampsia is one of the most common pregnancy disorders, and characterized by insufficient trophoblast invasion and placental inflammation. RNA sequencing was performed using placental tissues collected from PE patients and control healthy pregnant women. The results showed that OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2) was downregulated in placenta from PE patients.