No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE161329 | GEO | 2020/11/13
REPOSITORIES: GEO
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Project description:BackgroundSingle-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30?min co-incubation at 4?°C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures.ResultsHere, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30?min at 4?°C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets.ConclusionsWe demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30?min co-incubation at 4?°C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.
| S-EPMC7816397 | biostudies-literature
Project description:No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells
| PRJNA677895 | ENA
Project description:Although high-resolution single-particle cryo-electron microscopy (cryo-EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the preparation of suitable frozen-hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous - while still in the test tube - may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo-EM field.
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Project description:An automated microfluidic sample preparation multiplexer (SPM) has been developed and evaluated for Ebola virus detection. Metered air bubbles controlled by microvalves are used to improve bead-solution mixing thereby enhancing the hybridization of the target Ebola virus RNA with capture probes bound to the beads. The method uses thermally stable 4-formyl benzamide functionalized (4FB) magnetic beads rather than streptavidin coated beads with a high density of capture probes to improve the target capture efficiency. Exploiting an on-chip concentration protocol in the SPM and the single molecule detection capability of the antiresonant reflecting optical waveguide (ARROW) biosensor chip, a detection limit of 0.021pfu/mL for clinical samples is achieved without target amplification. This RNA target capture efficiency is two orders of magnitude higher than previous results using streptavidin beads and the limit of detection (LOD) improves 10×. The wide dynamic range of this technique covers the whole clinically applicable concentration range. In addition, the current sample preparation time is ~1h which is eight times faster than previous work. This multiplexed, miniaturized sample preparation microdevice establishes a key technology that intended to develop next generation point-of-care (POC) detection system.
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Project description:In MINUTE-ChIP, native chromatin is fragmented using Micrococcal nuclease, and subsequently blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Native MINUTE-ChIP is based on Mint-ChIP, developed by the Bernstein lab (van Galen et al., 2016; PMID: 26687680). We have introduce unique molecule (UMI) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTE-ChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. Here, we generate a standard curve for H3K27me3 and demonstrate that MINUTE-ChIP has a large linear dynamic range, thus MINUTE-ChIP quantitation is proportional to real quantities.
2019-06-21 | GSE133056 | GEO
Project description:Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.
| S-EPMC10684380 | biostudies-literature
Project description:In the urinary metabolomics for finding biomarkers in urine, owing to high concentrations of urea, for chromatography-based metabolomic analysis, urea needed to be degraded by urease. This urease pretreatment has been the key step of sample preparation for standard urinary metabolomics until today even for mass spectrometry-based analysis. The urease pretreatment involving incubation of urine with urease contradicts the concept of metabolome sampling, which should immediately arrest metabolic reactions to prevent alterations of a metabolite profile. Nonetheless, the impact of urease pretreatment has not been clearly elucidated yet. We found that activities of urease and endogenous urinary enzymes and metabolite contaminants from the urease preparations introduce artefacts into metabolite profiles, thus leading to misinterpretation.
| S-EPMC7077817 | biostudies-literature
Project description:Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses at the point-of-care is crucial for reducing disease transmission during the current pandemic and future flu seasons. To prepare for potential cocirculation of these two viruses, we report a valve-enabled, paper-based sample preparation device integrated with isothermal amplification for their simultaneous detection. The device incorporates (1) virus lysis and RNA enrichment, enabled by ball-based valves for sequential delivery of reagents with no pipet requirement, (2) reverse transcription loop-mediated isothermal amplification, carried out in a coffee mug, and (3) colorimetric detection. We have used the device for simultaneously detecting inactivated SARS-CoV-2 and influenza A H1N1 viruses in 50 min, with limits of detection at 2 and 6 genome equivalents, respectively. The device was further demonstrated to detect both viruses in environmental samples.
| S-EPMC8609915 | biostudies-literature
Project description:Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) Calibration Experiment
| PRJNA549856 | ENA
Project description:Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host's lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.
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