Low-input Samples Research Articles

Optimizing single cell proteomics using trapped ion mobility spectrometry for label-free experiments.

Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Furthermore, trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated in data-dependent acquisition mode (DDA-PASEF) allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating the ion flux in TIMS affects the overall performance of proteome profiling. However, the effect of TIMS settings on the analysis of low-input samples has been less investigated. Thus, we sought to optimize the conditions of TIMS with regard to ion accumulation/ramp times and ion mobility range for low-input samples. We observed that an ion accumulation time of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 V s cm-2 resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We used these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1116, and 1651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from a low number of cells was sufficient to delineate several essential metabolic pathways and the T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that such an approach could be applied to label-free analysis of single cells obtained from clinically relevant samples.

Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

Easy Hi-C: A Low-Input Method for Capturing Genome Organization.

Chromosome conformation capture technology and its derivatives have been widely used to study genome organization. Among them, Hi-C (chromosome conformation capture coupling with high-throughput sequencing) is popular in dissecting chromatin architecture on the genome-wide level. However, the intrinsic limitations prevent its application when it comes to rare samples. Here, we present easy Hi-C, a biotin-free technology that dramatically reduces DNA loss and is suitable for low-input samples.

Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0

BackgroundRNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue.ResultsWe developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance.ConclusionsrG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.

Effect of welding parameters on bead characteristics and mechanical properties of wire and arc additive manufactured inconel 718

In this study, a bi-directional, low-cost, gas metal arc welding (GMAW) based wire arc additive manufacturing (WAAM) setup is used to deposit aerospace-grade super-alloy IN718. The orthogonally designed experiments are carried out to optimize process parameters for bead geometry characteristics. Study result shows that with an increase in heat input, the bead parameters like bead width, reinforcement, dilution, penetration, and weld form factor are increasing, while it has a detrimental effect on weld shape factor and wetting angle. Microstructural examination reveals that at high heat input, coarser grain structure having average grain size of 72.29 µm is obtained while at low heat input, a finer grain structure with average grain size of 32.12 µm along with smaller amount of Laves phases are present. The XRD reveals NbC and TiC phases present in case of low heat input sample which enhances the mechanical properties of deposited material. The multilayer wall structures fabricated at high heat input showed more uniformity in shape with lesser waviness than that fabricated with low heat input. The tensile result of low heat input sample (775.82 MPa) shows higher strength than high heat input sample (741.07 MPa) which is because of smaller grain structure and higher hardness (278.11 HV) as obtained in low heat input sample as compared to high heat input sample (257.26 HV). The EBSD analysis also confirms highly textured surface, more grain boundary lengths and larger grain boundary orientation in low heat input sample imparted better mechanical properties.

Transcriptome-Wide Gene Expression Profiles from FFPE Materials Based on a Nuclease Protection Assay Reveals Significantly Different Patterns between Synovial Sarcomas and Morphologic Mimickers.

Simple SummaryThe following study aimed to validate and test the feasibility, reliability, technical applicability robustness, and reliability of a new commercially available transcriptome profiling assay, being performed on formalin-fixed, paraffin-embedded (FFPE) patient samples. The patients were suffering from either synovial sarcoma or spindle cell sarcoma, which are morphologically similar tumors, but differ in molecular characteristics. Transcriptome analysis of FFPE material is still challenging. However, it is the most available material in pathological routine and therefore valuable for translational research approaches. This new commercially available assay is based on a nuclease protection assay and has shown to be a feasible method for adequate transcriptome profiling with low sample input and therefore is suitable for further research of biomarkers. Background: Transcriptome profiling provides large data on tumor biology, which is particularly valuable in translational research and is becoming more and more important for clinical decision-making as well. RNA sequencing is considered to be the gold standard for this. However, FFPE material, as the most available material in routine pathology, has been an undefeatable obstacle for RNAseq. Extraction-free nuclease protection assays have the potential to be a reliable alternative method for large-scale expression profiling. The aim of this study was to validate and test the basic feasibility, technical applicability robustness, and reliability of the HTG transcriptome profiling (HTP) assay on clinical tumor samples. Methods: FFPE samples from 44 synovial sarcomas (SyS) and 20 spindle cell sarcomas (SpcS) were used. The HTP assay was performed on 10 µm thin FFPE slides. After nuclease protection in the HTG Edge Seq System, libraries were generated for sequencing on an Illumina NextSeq 500 platform. Fastq data were parsed and then analyzed by using the HTG analysis platform EdgeSeq REVEAL. Immunohistochemistry was performed to validate the expression of TLE1. Results: The technical application of the HTP Panel revealed robust and reliable results with 62 samples, and only 2 samples failed due to an incomplete digestion of gDNA. The analysis, performed at the analysis platform REVEAL, showed 5964 genes being significantly differentially expressed between SpcS and SyS. In particular, overexpression of the known marker TLE1 in synovial sarcoma could be recovered, which underlines the reliability of this system. Discussion: Transcriptome profiling gets more and more important for tumor research and diagnostics. Among other established technologies, the HTP Panel has shown to be a feasible method to get robust and reliable results. Thereby, this method needs very few sample-input by getting a success-rate of 96.88%, which indicates the upper average range, compared to other technologies working with FFPE tissue. Conclusion: The nuclease protection assay-based HTP Panel is a feasible method for adequate transcriptome profiling with low sample input and therefore is suitable for further research of biomarkers.

Optimized Repli-seq: improved DNA replication timing analysis by next-generation sequencing.

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type-specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

Abstract 3765: Epigenomics at the speed of sound: High throughput epigenomic mapping of low cell input samples using acoustic enhanced immunoprecipitation

Abstract Mapping the epigenetic landscape of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) is essential for understanding gene regulatory mechanisms during cell fate changes, disease onset, progression, as well as for optimizing therapeutic regime. In addition, these information hold immense potential for accurate identification of disease biomarkers, cell of origin, and targets for epigenetic therapy. However, existing chromatin immunoprecipitation sequencing (ChIP-Seq) workflows require significant cellular material and are very time-consuming, requiring cumbersome optimization. Moreover, cellular heterogeneity can influence the accuracy of results from the perspective of sample preparation efficiency and also population-biased effects. Both normal and malignant bone marrow are heterogeneous in cell type and stage of differentiation that leads to imprecise results with regard to epigenetic determinates.In our study, we implemented Covaris’ Adaptive Focused Acoustics® (AFA®) technology to miniaturize sample volumes for both chromatin shearing and immunoprecipitation in a 96-well format. This approach enables a standardized, robust, automatable, half-day workflow for both ChIP and Hi-ChIP applications. Additionally, we evaluated low input in vitro and in vivo samples, where we utilized a range of epitopes including major histone modifications (H3K27ac) and factors (CTCF). AFA was applied to flow-sorted stem and progenitor populations from individual mice. The low cell input capability permitted deconvolution of dynamic epigenetic changes in protein binding and chromatin structure during normal lineage specification of hematopoietic stem cells. Epigenetic remodeling of these sites were consistent with established gene programs essential for myeloid differentiation. Data were found to be in alignment with standard ChIP-seq and Hi-C datasets generated from similar cell populations.The simplified and significantly shortened (Hi)ChIP-seq processes showcased in this study will be highly beneficial for both research and clinical diagnostic applications owing to their requirements for using 96-well plate format and their ability to become integrated into automated workflows. Taken together, this technology and workflow represent a high throughput epigenetic system for small cell numbers of cells with a multitude of applications enabling laboratories focused on cell biology and translational research. Citation Format: Besmira Alija, Stephanie Braunstein, Jane Xu, Tom O'Hare, Angela Garding, Aaron Viny. Epigenomics at the speed of sound: High throughput epigenomic mapping of low cell input samples using acoustic enhanced immunoprecipitation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3765.

Abstract 540: Genome-wide circulating tumor DNA for monitoring treatment response and metastatic relapse in bladder cancer

Abstract Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (CX), is gold standard treatment in localized muscle-invasive bladder cancer (MIBC). About 45% of patients with MIBC develop metastatic relapse within 2 years after CX. The response rate to chemotherapy and immune checkpoint inhibitors (ICI) is relatively low, and biomarker tests for monitoring response are needed. Furthermore, biomarkers for early detection of minimal residual disease (MRD) after CX is needed to enable earlier treatment initiation. Tumor-informed detection of mutations in cell-free DNA (cfDNA) from peripheral blood has shown promising results in its ability to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied locally a whole-genome sequencing (WGS) approach to circulating tumor DNA (ctDNA) monitoring for improving ctDNA detection. Methods: A total of 140 MIBC patients undergoing NAC and CX were enrolled, including a test cohort (n=19) and a validation cohort (n=120). cfDNA was extracted from ~1mL plasma (n=1100) and procured from longitudinal plasma sampling during NAC (response measure), pre-cystectomy (response measure), post-surgery (relapse monitoring) and during immunotherapy (ICI treatment). WGS was applied to tumor/germline pairs (coverage >30x/20x) and plasma cfDNA (>20x) facilitating detection of genome wide genomic alterations and quantification of ctDNA using the MRDetect method. Results: We developed a personalized tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were then used for detecting and measuring the ctDNA levels in low-input blood samples by WGS. The assay sensitivity allowed for detection of tumor fractions down to 8*10-5. Furthermore, in our test cohort of 19 patients, we detected ctDNA after CX in 7 of 8 patients with clinical relapse (88% sensitivity) and detected no ctDNA in 11 of 11 patients with no clinical relapse (100% specificity). We observed a positive lead-time for MRD-based recurrence detection compared to CT-based reccurence detection (9 months on average). The full dataset is currently being processed and will be presented at the AACR 2022 meeting. Conclusions: For precision oncology, we need to develop quantitative and non-invasive methodologies to help tailor the treatments to individual patients and monitor them for further clinical decision-making. The results indicate the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring which could aid in clinical management of patients with bladder cancer. Citation Format: Iver Nordentoft, Karin Birkenkamp-Demtröder, Emil Christensen, Sunil Deochand, Dillon Maloney, Danielle Afterman, Tomer Lauterman, Noah Friedman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Sia Viborg, Mads Agerbæk, Jørgen Bjerggaard Jensen, Jonathan Rosenfeld, Ravi Kandasamy, Iman Tavassoly, Boris Oklander, Asaf Zviran, Lars Dyrskjøt. Genome-wide circulating tumor DNA for monitoring treatment response and metastatic relapse in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 540.

Abstract LB167: Functional extracellular vesicle profiling with nanochips for cancer diagnosis and monitoring

Abstract Currently, most cancer cases are diagnosed with local or distant metastases that lead to ~90% of deaths from cancer. Widely used radiographic imaging detects large-size tumors and often fails to detect changes in tumor burden. Genomics centered on gene alterations faces challenges in capturing instant status of malignancy, such as invasive/metastatic phenotypes. In addition, tissue biopsy is highly invasive, costly, and often infeasible to repeat. Thus, novel non-invasive markers and tests that complement current paradigms for early detection and accurate tracking of tumor dynamics are urgently needed to improve clinical decision making and treatment. Extracellular vesicles (EVs), including exosomes, are emerging as a new class of liquid biopsy for cancer diagnosis and monitoring response to treatment. Growing evidence indicates important functions of EVs in tumor development, invasion, and metastasis, including matrix remodeling via transporting matrix metalloproteases (MMPs). However, their clinical relevance remains largely undetermined, partially owing to the challenges in EV analysis. We recently report a nanoengineered lab-on-a-chip system capable of integrative functional phenotyping of tumor-associated EVs. A generalized, high-resolution colloidal inkjet printing method was developed to afford robust and widely adaptable manufacturing of 3D nanopatterned microdevices. Enabled by this new nanofabrication technique, we built an integrated chip for multiparametric analysis of exosome circulation level, subtype, and enzymolytic activity (ExoCLUE). With various cancer cell lines and isogenic cell models created by CRISPR/Cas9 editing, we demonstrated that this nanochip affords ultrasensitive and specific analysis detection of the expression and proteolytic activity of MMP14 on EVs to measure in vitro cell invasiveness. With the experimental and spontaneous mouse models of breast cancer metastasis, we showed that the ExoCLUE chip with low sample input enables longitudinal monitoring of in vivo tumor growth and metastasis in single mice. Lastly, the ExoCLUE was assessed for clinical analysis of plasma from two independent training (n = 30) and validation (n = 70) cohorts of breast cancer patients. The proteolytic activity of MMP14+ EVs showed good performance to detect the patients (AUC = 0.986 for the validation set). Combined with a machine learning algorithm, the chip-based integrative functional EV analysis enabled classification of the control, pre-malignant ductal carcinoma in situ, invasive ductal carcinoma, and locally metastatic IDC patients with a high accuracy of 92.9%. Lastly, we have scaled up the ExoCLUE system to a high-throughput nanoarray platform for highly multiplexed functional EV profiling. Overall, our nanochip technology could provide a useful liquid biopsy tool to improve animal studies and clinical diagnosis of cancer. Citation Format: Yong Zeng, Shibo Chen, Yunjie Wen, Liang Xu. Functional extracellular vesicle profiling with nanochips for cancer diagnosis and monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB167.

Analytical Performance Evaluation of New DESI Enhancements for Targeted Drug Quantification in Tissue Sections.

Desorption/ionization (DI)-mass spectrometric (MS) methods offer considerable advantages of rapidity and low-sample input for the analysis of solid biological matrices such as tissue sections. The concept of desorption electrospray ionization (DESI) offers the possibility to ionize compounds from solid surfaces at atmospheric pressure, without the addition of organic compounds to initiate desorption. However, severe drawbacks from former DESI hardware stability made the development of assays for drug quantification difficult. In the present study, the potential of new prototype source setups (High Performance DESI Sprayer and Heated Transfer Line) for the development of drug quantification assays in tissue sections was evaluated. It was demonstrated that following dedicated optimization, new DESI XS enhancements present promising options regarding targeted quantitative analyses. As a model compound for these developments, ulixertinib, an inhibitor of extracellular signal-regulated kinase (ERK) 1 and 2 was used.

Investigating chromatin accessibility during development and differentiation by ATAC-sequencing to guide the identification of cis-regulatory elements.

Mapping accessible chromatin across time scales can give insights into its dynamic nature, for example during cellular differentiation and tissue or organism development. Analysis of such data can be utilised to identify functional cis-regulatory elements (CRE) and transcription factor binding sites and, when combined with transcriptomics, can reveal gene regulatory networks (GRNs) of expressed genes. Chromatin accessibility mapping is a powerful approach and can be performed using ATAC-sequencing (ATAC-seq), whereby Tn5 transposase inserts sequencing adaptors into genomic DNA to identify differentially accessible regions of chromatin in different cell populations. It requires low sample input and can be performed and analysed relatively quickly compared with other methods. The data generated from ATAC-seq, along with other genomic approaches, can help uncover chromatin packaging and potential cis-regulatory elements that may be responsible for gene expression. Here, we describe the ATAC-seq approach and give examples from mainly vertebrate embryonic development, where such datasets have identified the highly dynamic nature of chromatin, with differing landscapes between cellular precursors for different lineages.

Zwitter-ionic monolith-based spintip column coupled with Evosep One liquid chromatography for high-throughput proteomic analysis

A high-throughput proteomic workflow with good sensitivity and reproducibility is highly demanding for proteomic studies of large clinical cohorts. We present a workflow that seamlessly integrates the zwitter-ionic monolith-based spintip (ZIM-Tip) with the Evosep One liquid chromatography system to address this challenge. Disposable ZIM-Tips were prepared with satisfying permeability based on photo-initiated free radical polymerization. Sample preparation steps, including ion-exchange-based protein concentration, reduction, alkylation, and enzymatic digestion, were processed on the ZIM-Tips in 2 h with about 10% sample loss. The peptides recovered from ZIM-Tips were directly loaded on Evotips for desalting and proteomic data acquisition. In one-hour high performance liquid chromatography-MS/MS run, more than 4000 proteins were consistently identified from 1 µg of cell lysate using timsTOF Pro-mass spectrometer in data-dependent acquisition mode (DDA). At least 20 samples with protein amount of 1 µg could be processed each day. Good intra- and inter-day precision in quantification were demonstrated with median coefficient of variation (CV) values of less than 20% and 30%, respectively. The average Pearson correlation coefficients of each two sets of samples are 0.934 and 0.901, respectively. Collectively, the ZIM-Tip technology offers an useful solution for clinical cohort studies with demand for large sample amounts and low sample input while maintaining in-depth proteome coverage.

A Partially Multiplexed HIV Drug Resistance (HIVDR) Assay for Monitoring HIVDR Mutations of the Protease, Reverse-Transcriptase (PRRT), and Integrase (INT).

ABSTRACTAs dolutegravir (DTG)-containing HIV regimens are scaled up globally, monitoring for HIV drug resistance (HIVDR) will become increasingly important. We designed a partially multiplexed HIVDR assay using Sanger sequencing technology to monitor HIVDR mutations in the protease, reverse-transcriptase (PRRT), and integrase (INT). A total of 213 clinical and analytical plasma and dried blood spot (DBS) samples were used in the evaluation. The assay detected a wide range of known HIV-1 subtypes and circulating recombinant forms (CRFs) of group M from 139 samples. INT accuracy showed that the average nucleotide (nt) sequence concordance was 99.8% for 75 plasma samples and 99.5% for 11 DBS samples compared with the reference sequences. The PRRT accuracy also demonstrated the average nucleotide sequence concordance was 99.5% for 57 plasma samples and 99.2% for 33 DBS samples. The major PRRT and INT DR mutations of all samples tested were concordant with those of the reference sequences using the Stanford HIV database (db). Amplification sensitivity of samples with viral load (VL) >5000 copies/mL showed plasma exceeded 95% of positivity, and DBS exceeded 90% for PRRT and INT. Samples with VL (1000 to 5000 copies/mL) showed plasma exceeded 90%, and DBS reached 88% positivity for PRRT and INT. Assay precision and reproducibility showed >99% nucleotide sequence concordance in each set of replicates for PRRT and INT. In conclusion, this HIVDR assay met WHO HIVDR assay performance criteria for surveillance, worked for plasma and DBS, used minimal sample volume, was sensitive, and was a potentially cost-effective tool to monitor HIVDR mutations in PRRT and INT.IMPORTANCE This HIVDR genotyping assay works for both plasma and DBS samples, requires low sample input, and is sensitive. This assay has the potential to be a user-friendly and cost-effective HIVDR assay because of its partially multiplexed design. Application of this genotyping assay will help HIVDR monitoring in HIV high-burdened countries using a DGT-based HIV drug regimen recommended by the U.S. President's Emergency Plan for AIDS Relief and the WHO.

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering.

SUMMARYSingle-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells, with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes. A record of this paper’s transparent peer review process is included in the supplemental information.

SEEMLIS: a flexible semi-automated method for enrichment of methylated DNA from low-input samples

BackgroundDNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS.ResultsWe show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity.ConclusionsSEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

Hydrop enables droplet-based single-cell ATAC-seq and single-cell RNA-seq using dissolvable hydrogel beads.

Single-cell RNA-seq and single-cell assay for transposase-accessible chromatin (ATAC-seq) technologies are used extensively to create cell type atlases for a wide range of organisms, tissues, and disease processes. To increase the scale of these atlases, lower the cost and pave the way for more specialized multiome assays, custom droplet microfluidics may provide solutions complementary to commercial setups. We developed HyDrop, a flexible and open-source droplet microfluidic platform encompassing three protocols. The first protocol involves creating dissolvable hydrogel beads with custom oligos that can be released in the droplets. In the second protocol, we demonstrate the use of these beads for HyDrop-ATAC, a low-cost noncommercial scATAC-seq protocol in droplets. After validating HyDrop-ATAC, we applied it to flash-frozen mouse cortex and generated 7996 high-quality single-cell chromatin accessibility profiles in a single run. In the third protocol, we adapt both the reaction chemistry and the capture sequence of the barcoded hydrogel bead to capture mRNA, and demonstrate a significant improvement in throughput and sensitivity compared to previous open-source droplet-based scRNA-seq assays (Drop-seq and inDrop). Similarly, we applied HyDrop-RNA to flash-frozen mouse cortex and generated 9508 single-cell transcriptomes closely matching reference single-cell gene expression data. Finally, we leveraged HyDrop-RNA's high capture rate to analyze a small population of fluorescence-activated cell sorted neurons from the Drosophila brain, confirming the protocol's applicability to low input samples and small cells. HyDrop is currently capable of generating single-cell data in high throughput and at a reduced cost compared to commercial methods, and we envision that HyDrop can be further developed to be compatible with novel (multi) omics protocols.

Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repression of cytosolic translation after TUFM perturbation. We show the assay is applicable to small amounts of primary tissue samples with low protein synthesis rates, including snap-frozen tissues and immune cells from an individual's blood draw. We showed its feasibility to characterize the personalized immuno-translatome. Our analyses revealed that thousands of genes show lower translation efficiency in monocytes compared with lymphocytes, and identified thousands of translated noncanonical open reading frames (ORFs). Altogether, our RNase footprinting approach opens an avenue to assay transcriptome-wide translation using low-input samples from a wide range of physiological conditions.

Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition.

Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues.

Systematic comparative analysis of strand-specific RNA-seq library preparation methods for low input samples

Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increases the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies—Swift RNA and Swift Rapid RNA, presently offered by Integrated DNA Technologies (IDT) —alongside the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark gene expression in these kits, at input quantities ranging between 10 to 500 ng. We found normalized read counts between all treatment groups to be in high agreement. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits offer shorter workflow times enabled by their patented Adaptase technology. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways directly attributable to input mRNA amount.