Project description:In this study, we performed a comparative analysis of gut microbiota composition and gut microbiome-derived bacterial extracellular vesicles (bEVs) isolated from patients with solid tumours and healthy controls. After isolating bEVs from the faeces of solid tumour patients and healthy controls, we performed spectrometry analysis of their proteomes and next-generation sequencing (NGS) of the 16S gene. We also investigated the gut microbiomes of faeces from patientsand controls using 16S rRNA sequencing. Machine learning was used to classify the samples into patients and controls based on their bEVs and faecal microbiomes.
Project description:This study aims to investigate the role of Lysine Methyltransferase 5A (KMT5A, also known as SETD8) in CD8+ T cell biology. CD8+ T cells were isolated from healthy volunteer PBMCs using magnetic bead-based negative selection. Isolated cells were subjected to CRISPR-Cas9 mediated gene editing targeting KMT5A (KMT5A-KO group) or a non-targeting control guide RNA (control group, CTRL) via ribonucleoprotein (RNP) complex electroporation. Cells were harvested at 72 hours post-electroporation for RNA extraction. RNA sequencing (RNA-seq) libraries were constructed using the Illumina TruSeq Stranded mRNA kit and sequenced on an Illumina platform to generate paired-end reads. Transcriptome profiles of KMT5A-KO CD8+ T cells were compared to those of CTRL cells to identify differentially expressed genes (DEGs) and elucidate potential pathways regulated by KMT5A in this cell type.
Project description:We compared the microbiota of paired mouse caecal contents and faeces by applying a multi-omic approach, including 16S rDNA sequencing, shotgun metagenomics, and shotgun metaproteomics. The aim of the study was to verify whether faecal samples are a reliable proxy for the mouse colonic luminal microbiota, as well as to identify changes in taxonomy and functional activity between caecal and faecal microbial communities, which have to be carefully considered when using stool as sample for mouse gut microbiota investigations.
Project description:Six- to eight-week old CD1 male mice were sleep-deprived for 24 hours by placing them in a small platform in a water tank. Their ileum samples were harvested every 4 hours on the next day (time point: ZT1,ZT5,ZT9,ZT13,ZT17,ZT21). The ileum samples were subjected to 16s rDNA sequencing. Control group (C1,C5,C9,C13,C17,C21, n=36). SD group (SD1,SD5,SD9,SD13,SD17,SD21, n=36).
Project description:Human stool samples were collected, microbial cells were harvested and subjected to protein extraction using different protocols for metaproteomics analyses. The protein extraction methods differ from the usage of different lysis buffer (SDS, urea and commercially available B-Per reagent), bead beating, ultrasonication and heating. The impacts of different protein extraction methods on metaproteomics results were evaluated with multiple criteria including protein yields, peptide identifications, taxonomic compositions and functional compositions.
Project description:Study investigates the biological role of small RNAs present in aqueous humor in patients with primary open-angle glaucoma, pseudoexfoliative glaucoma, and cataract, with the aim of identifying molecular biomarkers and improving understanding of disease mechanisms. Aqueous humor samples were collected during ophthalmic surgery, followed by RNA extraction from the cell-free supernatant using a commercial kit designed for low-input RNA. Small RNA sequencing libraries were prepared using adapter ligation with Unique Molecular Identifiers, reverse transcription, PCR amplification, and magnetic bead purification, and library quality was assessed using a microfluidics-based system. The pooled libraries were sequenced using paired-end sequencing on an Illumina NextSeq platform, and the resulting FASTQ files were used for downstream bioinformatic and machine learning analyses integrating molecular, clinical, and imaging data.
Project description:We profiled the DNA methylation of saliva cell types, to develop a tool for epidemiologic studies. Saliva was collected from 22 children, 21 participants with samples usable for DNA methylation, and sorted into immune and epithelial cells, using size exclusion filtration and magnetic bead sorting. DNA methylation was measured using the Illumina MethylationEPIC BeadChip. Saliva immune and epithelial cells have distinct DNA methylation profiles, which can influence whole saliva epidemiologic measures.
Project description:Optimisation of a bead-beating procedure for simultaneous extraction of bacterial and fungal DNA from pig faeces and liquid feed for 16S and ITS2 rDNA amplicon sequencing