Bead-beating method development and optimisation
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ABSTRACT:
PROVIDER: PRJEB49004 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Fungiform papillae (FP) are visible protrusions on the anterior tongue surface that contain taste buds, their nerves, and capillaries, epithelial cells, stromal cells, and immune-surveilling cells. As FP are easily biopsied in a minimally invasive procedure and have been shown to regrow, we compared three different mechanical methods of FP protein extraction and found that mechanical disruption of FP under liquid nitrogen or bead beating were more efficient than mincing in terms of yield and proteomic profile. A successful protocol to extract RNA from individual FP using bead beating was employed for transcriptomic analysis by RNA sequencing and microarray. Relative to female FP, male FP proteomes and transcriptomes showed predominant expression of Y chromosome-encoded Y antigens, such as EIF1AY, DDX3Y, SMCY, NLGN4Y, UTY, and PCDH11Y, validating our findings. We further found significantly higher levels of Human Leucocyte Antigen (HLA)-associated transcripts and HLA-A protein levels in males than in females. Several long intergenic non-coding (LINC) RNAs exhibit sex-specific expression, in particular LIN02888 with unknown function is more highly expressed in female relative to male FP. Transcripts in the interferon response pathway a key element of the innate immune system's defense against pathogens exhibits differential regulation between the sexes. This indicates that FP biopsy is a minimally invasive method to obtain human tissue in order to study, for example, age-related and sex specific changes of interest, while allowing for dynamic investigation of interindividual differences over time in the context of both health and disease.
2021-12-14 | GSE190832 | GEO
Project description:Fungiform papillae (FP) are visible protrusions on the anterior tongue surface that contain taste buds, their nerves, and capillaries, epithelial cells, stromal cells, and immune-surveilling cells. As FP are easily biopsied in a minimally invasive procedure and have been shown to regrow, we compared three different mechanical methods of FP protein extraction and found that mechanical disruption of FP under liquid nitrogen or bead beating were more efficient than mincing in terms of yield and proteomic profile. A successful protocol to extract RNA from individual FP using bead beating was employed for transcriptomic analysis by RNA sequencing and microarray. Relative to female FP, male FP proteomes and transcriptomes showed predominant expression of Y chromosome-encoded Y antigens, such as EIF1AY, DDX3Y, SMCY, NLGN4Y, UTY, and PCDH11Y, validating our findings. We further found significantly higher levels of Human Leucocyte Antigen (HLA)-associated transcripts and HLA-A protein levels in males than in females. Several long intergenic non-coding (LINC) RNAs exhibit sex-specific expression, in particular LIN02888 with unknown function is more highly expressed in female relative to male FP. Transcripts in the interferon response pathway a key element of the innate immune system’s defense against pathogens exhibits differential regulation between the sexes. This indicates that FP biopsy is a minimally invasive method to obtain human tissue in order to study, for example, age-related and sex specific changes of interest, while allowing for dynamic investigation of interindividual differences over time in the context of both health and disease
2023-12-25 | GSE221229 | GEO
Project description:The goal of this study was to optimize protein extraction methods to study root-associated bacteria in Arabidopsis. For this we inoculated Arabidopsis seedlings grown in agar plates with a synthetic community (SynSom) composed of four different strains (Variovorax paradoxus, Arthrobacter sp, Agrobacterium sp. and Pseudomonas sp.. Twelve days after inoculation we extracted proteins from the roots using six different protein extraction methods each in triplicates. These methods were a combination of different extraction buffers (SDS or Triton-based) and mechanical disruption methods (bead-beating, N2 grinding, glass homogenizer and freeze-thaw cycles) We found that bead-beating the roots with lysing matrix E in SDT lysis buffer yielded the highest numbers of microbial protein identification and enhanced the detection of proteins derived from gram positive bacteria.
2022-09-08 | PXD026330 | Pride
Project description:A specific discovery proteomic protocol for intervertebral discs relies on an efficient protein extraction using the bead beating homogenizing method with stainless steel grinding balls coupled with a customized lysis buffer, and followed by the protein detection with specific mass spectrometry setting.
2019-09-11 | PXD015033 | Pride
Project description:16S rDNA Illumina analysis of mouse faeces using magnetic-bead based extraction
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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.
2017-07-17 | PXD005969 | Pride
Project description:The goal of this study was to optimize protein extraction methods to study root-associated bacteria in maize. For this we inoculated sterile maize plants with a synthetic community composed of seven different bacteria (Ben Niu et al. PNAS 2017, vol 114, n 12). Then, we extracted proteins from maize roots using eight different protein extraction methods in triplicates. These methods were a combination of different extraction buffers (SDS or Triton-based) and mechanical disruption methods (bead-beating, N2 grinding, glass homogenizer and freeze-thaw cycles). We found that vortexing maize roots with glass beads in PBS yielded the highest numbers of microbial protein identification.
2022-09-08 | PXD026369 | Pride
Project description:Metaproteomic studies of full-scale activated sludge systems require reproducible protein extraction methods. A systematic evaluation of three different extractions protocols, each in combination with three different methods of cell lysis, and a commercial kit were evaluated. Criteria used for comparison of each method included the extracted protein concentration and the number of identified proteins and peptides as well as their phylogenetic, cell localization and functional distribution and quantitative reproducibility. Furthermore, the advantage of using specific metagenomes and a 2-step database approach was illustrated. The results recommend a protocol for protein extraction from activated sludge based on the protein extraction reagent B-Per and bead beating.
2014-08-15 | PXD000862 | Pride
Project description:Spatial transcriptomics technologies that can quantify gene expression in space are transforming contemporary biology research. Some of such methods use spatially barcoded bead arrays that are optically sequenced by a microscopy setup to detect bead barcodes in space which can be consecutively matched to cell barcodes from the respective single cell sequencing experiment. To have good quality barcodes and a high number of barcode matches in space, robust and efficient computational pipelines are needed to process raw microscopy images and call the bases of bead barcodes accurately. Here, we present Optocoder, a computational pipeline that takes raw optical sequencing microscopy images as input and outputs bead barcodes in space. Optocoder efficiently aligns images, detects beads, and corrects for confounding factors of the fluorescence signal such as crosstalk and phasing before base calling. Furthermore, we implement a machine learning pipeline that is trained using the signal from the beads that match to illumina barcodes in order to predict non-matching bead barcodes which can boost up the number of barcode matches. We benchmark Optocoder using data from an in-house spatial transcriptomics platform as well as data from the Slide-seq method and we show that it can efficiently process both datasets with minimal modification.
2022-06-24 | GSE193472 | GEO
Project description:Setup and optimisation of a high throughput pipeline for ChIPseq. The protocol used is ChIP carried out using the Agilent Bravo robot with subsequent Ilumina sequencing library preparation.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2013-02-12 | E-ERAD-77 | biostudies-arrayexpress