Transcriptome sequencing (RNA-seq) is a powerful technology for gene expression profiling. Selection of optimal parameters for cDNA library generation is crucial for acquisition of high-quality data. In this study, we investigate the impact of the amount of RNA and the number of PCR cycles used for sample amplification on the rate of PCR duplication and, in consequence, on the RNA-seq data quality. For broader applicability, we sequenced the data on four short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. The native Illumina libraries were converted for sequencing on AVITI and G4 to assess the effect of library conversion, containing additional PCR cycles. We find that the rate of PCR duplicates depends on the combined effect of RNA input material and the number of PCR cycles used for amplification. For input amounts lower than 125 ng, 34–96

Rigor, reproducibility, and transparency (RR&T) are essential components of all scientific pursuits. Shared research resources, also known as core facilities, are on the frontlines of ensuring robust RR&T practices. The Association of Biomolecular Resource Facilities Committee on Core Rigor and Reproducibility conducted a follow-up survey 4 years after the initial 2017 survey to determine if core facilities have seen a positive impact of new RR&T initiatives (including guidance from the National Institutes of Health, new scientific journal requirements on transparency and data provenance, and educational tools from professional organizations). While there were fewer participants in the most recent survey, the respondents' opinions on the role of core facilities and level of best practices adoption remained the same. Overall, the respondents agreed that procedures should be implemented by core facilities to ensure scientific RR&T. They also indicated that there is a strong correlation between institutions that emphasize RR&T and core customers using this expertise in grant applications and publications. The survey also assessed the impact of the COVID-19 pandemic on core operations and RR&T. The answers to these pandemic-related questions revealed that many of the strategies aimed at increasing efficiencies are also best practices related to RR&T, including the development of standard operating procedures, supply chain management, and cross training. Given the consistent and compelling awareness of the importance of RR&T expressed by core directors in 2017 and 2021 contrasted with the lack of apparent improvements over this time period, the authors recommend an adoption of RR&T statements by all core laboratories. Adhering to the RR&T guidelines will result in more efficient training, better compliance, and improved experimental approaches empowering cores to become "rigor champions."

Although messenger RNA (mRNA) is the focus of much RNA research, it constitutes a relatively small fraction of total RNA in a cell. Most cellular RNA is ribosomal RNA (rRNA) and removal of this RNA is is desirable in many RNA-Seq studies in order to maximize capacity of the sequencing instrument and reduce costs. We have employed the CRISPR Cas9 double-stranded DNA endonuclease to develop a different method of rRNA removal for RNA-Seq studies. Here we show data from the CRISPR treatment of rRNA sequences from RNA-Seq libraries prepared from three different bacteria and Homo sapiens.

Prolonged obesity is associated with blunted feeding and thermogenic autonomic responses to leptin, but cardiovascular responses to leptin are maintained. This state of selective leptin resistance is, therefore, proposed to contribute to the pathogenesis and maintenance of obesity-associated hypertension. Cells of the arcuate nucleus of the hypothalamus detect leptin, and although the cellular and molecular mechanisms remain unclear, altered arcuate nucleus biology is hypothesized to contribute to selective leptin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD) or chow from 8 to 18 weeks of age, as this paradigm models selective leptin resistance. Nuclei were then isolated from arcuate nucleus for single-nucleus RNA sequencing. HFD caused expected gains in adiposity and circulating leptin. Twenty-three unique cell-type clusters were identified, and Ingenuity Pathway Analysis was used to explore changes in gene expression patterns due to chronic HFD within each cluster. Notably, gene expression signatures related to leptin signaling exhibited suppression predominantly in neurons identified as the Agouti-related peptide (Agrp) subtype. Ingenuity Pathway Analysis results were also consistent with alterations in CREB (cAMP response element-binding protein) signaling in Agrp neurons after HFD, and reduced phosphorylated CREB was confirmed in arcuate nucleus after prolonged HFD by capillary electrophoresis-based Western blotting. These findings support the concept that prolonged HFD-induced obesity is associated with selective changes in Agrp neuron biology, possibly secondary to altered CREB signaling.

This work focusses on the application of high throughput workflows for proteome and phosphoproteome profiling followed by fast gradient liquid chromatography (LC) coupled to data-independent acquisition (DIA) mass spectrometry analysis. Automation of sample preparation increased throughput and reproducibility covering all steps from protein extract to mass spectrometry analysis allowing for parallel processing of up-to 96 samples in less than 6 hours (excluding digest time). We show the adaptation of the sample preparation methods including protein capture, clean-up, and on-bead digestion as well as phosphopeptide enrichment for use in a magnetic handling stations (Thermo Scientific King Fisher Flex) allowing for semi-automated sample processing. The methods are readily transferable to a range of liquid handling robots with magnetic handling stations (capabilities). Efficient protein isolation from wide range of tissues was achieved using 5% SDS followed by aggregation-based protein capture method (PAC) on MagReSyn® Amine magnetic microparticles allowing for efficient contaminant removal and on-bead digestion. For phosphoproteome profiling the PAC-generated peptides were further processed using MagReSyn® Ti-IMAC HP magnetic microparticles. All steps except for plate aliquoting and desalting were performed using a magnetic handling station that allowed for automated processing of up-to 96 samples in parallel. 500 ng peptides or enriched phosphopeptides were loaded directly on Evotips and further analysed using 21-minute gradients on Evosep LC coupled to Orbitrap Exploris equipped with FAIMS source allowing for up-to 60 samples per day to be analysed. Over 8,000 proteins and 22,000 unique phospho-peptides were quantified across the 12 tissues in a total mass spectrometry time of 28 hours.

Unique molecular identifiers (UMIs) have become a vital aspect of next-generation sequencing and are used for preserving quantitative information by removing PCR/sequencing errors and duplicates. Here, we integrated UMIs with our SMART® technology to detect genuine low-frequency events in full-length variable regions of BCR and TCR genes. The results showed detection sensitivity of rare clones at 0.001% and reproducibility for low amounts of total RNA (10 ng). Our technology can be used to observe clonal selection and hypermutation events in rare clonotypes found in blood and tumor tissues.

Protein phosphorylation is the most common mechanism of regulating protein function. With an expanding phosphoproteome of known functional pathways, much interest now turns towards targeted quantitative analyses based on candidates earlier identified in discovery screens for detailed comparison of differential phosphorylation and linking with genomic evidence of mutagenesis. Even complex tissue samples can be probed selectively for phosphopeptides of interest by devoting instrument sensitivity and sampling speed to a subset of relevant targets for peptide quantification. Here, we screen for phosphoproteins in the AKT/mTOR pathway using a new targeted sample prep approach (SureQuant). This approach provides a simple, robust method to multiplex immunoprecipitation and mass spec sample prep for multiple phosphopeptides simultaneously: this particular kit is used to identify and quantify 30 unique peptides from 10 phosphorylated proteins and can be used in conjunction with genomic analyses. This panel covers significant proteins throughout the AKT-mTOR signaling pathway from human clinical samples. Using this approach, we enriched for ∼300 proteins and ∼1k peptides from both cell lysates and tissues in untargeted runs. Most of the 10 AKT/mTOR targets were successfully identified from frozen tissue and lysates. Identification of a couple of targets in FFPE samples is encouraging and an indication of sensitivity even in these challenging samples. Quantitation of these targets is being evaluated in ongoing efforts. With this technology, we identify peptide targets from both cell lysates and frozen tissues, and additionally demonstrate viability for some of these target phosphoproteins in FFPE tissue, laying the foundation for connecting these observations with genetic screening. This new tool adds a critical 'next step' for phosphoproteomics approaches previously limited to qualitative screening and is portable to analogous protein targets in other research areas.

Shared research resource facilities, also known as core laboratories (Cores), are responsible for generating a significant and growing portion of the research data in academic biomedical research institutions. Cores represent a central repository for institutional knowledge management, with deep expertise in the strengths and limitations of technology and its applications. They inherently support transparency and scientific reproducibility by protecting against cognitive bias in research design and data analysis, and they have institutional responsibility for the conduct of research (research ethics, regulatory compliance, and financial accountability) performed in their Cores. The Association of Biomolecular Resource Facilities (ABRF) is a FASEB-member scientific society whose members are scientists and administrators that manage or support Cores. The ABRF Research Groups (RGs), representing expertise for an array of cutting-edge and established technology platforms, perform multicenter research studies to determine and communicate best practices and community-based standards. This review provides a summary of the contributions of the ABRF RGs to promote scientific rigor and reproducibility in Cores from the published literature, ABRF meetings, and ABRF RGs communications.

Desorption electrospray ionization (DESI) mass spectrometry (MS) is an ambient ionization source with a wide array of applications from high-throughput screening to molecular imaging. In this study, our objective was to design a novel sprayer using electro-flow focusing and examine if tighter focused beam can perform a higher spatial resolution DESI imaging. DESI imaging mass spectrometry (MS) is performed by obtaining a pixel-by-pixel mass spectrum of a sample by impinging focused electrospray droplets on a confined point, such as pixel. Thus, the spatial resolution or pixel size of DESI imaging is primarily defined by the area of the electrospray impact on the surface. A stable and highly focused electrospray is a prerequisite for a high spatial resolution DESI imaging experiment. An imaging sprayer should be capable of generating a robust MS signal from a few dozen microns sample area - from the first pixel to the last millionth pixel. To briefly summarize our results, a novel DESI sprayer was designed based on precision machined ion-key emitters (Waters Corporation). Under the optimal conditions, DESI solvent guided through the orifice by a sheathing nebulization gas was capable of creating a highly concentric and stable flow stream of < 20 µm in diameter. The stability, as well as, the size of the DESI spray was related to solvent flow, high voltage, and nebulization gas flow. DESI imaging beam was optimized by using shadowgraph imaging of electrospray plume. DESI ion source was interfaced with a quadrupole time-of-flight mass spectrometer (SYNAPT G2-XS, Waters Corporation). DESI solvent (95-98% methanol) was regulated using a binary LC pump (ACQUITY UPLC M-Class, Waters Corporation). In conclusion, the novel sprayer with electro-flow focusing technique that combined electrospray and flow focusing, was capable of high-resolution DESI imaging of metabolites and lipids in rat brain and chicken liver sections below 50 microns.

As nanopore sequencing technology matures, short read Next Generation Sequencing (NGS) applications can be supplemented. De novo transcript assembly, currently done at scale with short reads, could be replaced since the ability to sequence full length cDNA may increase detection of splice variants and fusion transcripts. Here, we present rough analyses of cDNA sequencing data on the MinION and PromethION as well as Qubit measurements and Fragment Analyzer traces. We compare this data against standard benchmarks of other NGS.

The ability to perform genome editing has expanded tremendously in recent years and has been facilitated by the development of systems based on clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) that allow targeted cleavage of genomic loci. We have tested the use of FlashGel™ DNA Cassettes as tools to monitor the results of several electrophoretic separation steps that maybe involved in the CRISPR/Cas9 workflow: mismatch cleavage assays, single guide RNA (sgRNA) screening, and single stranded DNA (ssDNA) production. Mismatch cleavage assays are a quick and inexpensive tool to evaluate the efficiency of editing. FlashGel™ DNA Cassettes allowed rapid (five minutes) separation of cleavage products with high sensitivity. Separation performance was confirmed using control reagents from seven commercially available mismatch cleavage kits. Testing additionally showed that FlashGel™ DNA Cassettes worked well for rapid separation of cleavage products generated using control reagents from two sgRNA screening kits. ssDNA is becoming more widely used as a donor for knock-in CRISPR/Cas9 experiments involving homology directed repair. Testing in conjunction with control reagents from a commercially available ssDNA production system showed that FlashGel™ DNA Cassettes allowed monitoring of ssDNA generation and gave the added benefit of being able to distinguish ssDNA and dsDNA based upon observation of the color of the bands. The use of FlashGel™ DNA Cassettes in gene editing workflows offers increased separation speed and detection sensitivity relative to separations using conventional agarose gel formats.

Using droplet sequencing and full-length mRNA information in parallel has become an emerging requirement to help generate and better understand rich single-cell datasets. To address this need, we developed the SMART-Seq® Single Cell Kit (SSsc) using new chemistry with unparalleled sensitivity and a highly scalable and easily automatable workflow. These features make SSsc chemistry extremely useful for difficult cells-e.g., clinical research samples that often have very low RNA content-making it ideal for highly detailed characterization of precious samples.

Isolating single cells at high-throughput levels and obtaining their full-length transcript information has become critical to the scientific community to generate rich single-cell datasets. We automated SMART-Seq® chemistry on the ICELL8® cx Single-Cell System to address this need and show this workflow provides useful information that end-capture technologies cannot. We present performance data showing that capturing junction and spanning reads with this automated, full-length mRNA-seq method enables confident and robust identification of gene fusions in a breast cancer tumor cell line.

Our novel, enzymatic DNA synthesis (EDS) technology was made through engineering of three critical components: a DNA polymerase, a solid support, and reversible terminators. Terminal deoxynucleotidyl-Transferase (TdT), which performs poly-nucleotide additions in a template-free manner, was engineered for improved expression, stability, and the ability to incorporate non-natural deoxynucleotide triphosphate bases. Novel surface chemistries have been developed to allow TdT to incorporate DNA bases on paramagnetic beads, resins, and glass as solid supports for EDS. Synthesis is enabled by the use of 3â€-O-blocked reversible terminators, which suppresses poly-nucleotide addition by TdT until the 3'OH group on the extended polymer is deprotected. Here, we describe the development and key functionality of a benchtop EDS system for lab use. Unlike phosphoramidite synthesis, EDS produces DNA in the 'biological' orientation (i.e. 5'->3'), with an intact 5'-phosphate group. EDS provides a scalable synthesis system that eliminates the need for solvents, like acetonitrile, minimizing organic waste and decreasing research's carbon footprint, allowing users to make oligos without the need for specialized training and delivering oligos for iterative experiments without having to wait for a centralized oligo manufacturer. Current data indicates that purity levels comparable to those of existing technologies can be achieved, with an average per-position error rate <1%. For oligos up to 280nt long, we have demonstrated a cycle efficiency of 99.4%, which is greater than or equivalent to that of standard phosphoramidite synthesis. Using PCR, qPCR, dPCR, and sequencing assays we have demonstrated comparable performance to the same sequences synthesized using conventional chemistry. EDS is a disruptive technology that enables simple, robust systems capable of supporting same-day production of high quality, custom oligos. On-demand access to oligos revolutionize genomics research.

Biotin labeling in combination with LC-MS/MS has been widely applied in large-scale analysis of protein post-translational modifications, cell surface proteins, protein-protein interactions, and protein subcellular localization. Direct identification of protein biotinylation sites are still challenging due to the low recovery of biotinylated peptides using conventional streptavidin/avidin-based purification methods. It has been found that anti-biotin antibody is a better capture reagent for biotinylated peptides compared to streptavidin/avidin. In this study, we established an immunoaffinity enrichment method using a monoclonal anti-biotin antibody and compared it to the published approaches from two papers using polyclonal antibodies from various vendors. We then demonstrated our enrichment method by applying it to characterization of protein biotinylation sites from proximity labeling studies in living cells.

Availability of biological material is often a limiting factor in generating robust, multi-omics datasets. Additionally, regional tissue heterogeneity obscures the ability to directly compare adjacent regions. This is particularly true of mass-spectrometry based metabolomics where analytes can't be amplified. Milligram quantities of tissue are needed to extract sufficient quantities of metabolites to detect important, lower abundant metabolites. Researchers often pool several individual samples or compare datasets from different animals for RNAseq and metabolomics, lowering experimental power and creating batch effects. We compared RNA quality and quantity, RNAseq expression profiling, and dispersion metrics among mouse liver samples 2 days post injection with Lymphocytic choriomeningitis virus (LCMV) or vehicle (VEH). The LCMV model was selected to provide high biological contrast with which to assess RNAseq results as a function of extraction method. Frozen liver samples were pulverized in liquid nitrogen and split into 2 aliquots, one for RNA extraction (RNA) and one for sequential metabolite extraction (80% MeOH) followed by RNA extraction (metRNA). This allowed for intra-individual correlations between RNA and metRNA. RNA quantity and integrity measurements were not statistically significant between metRNA and RNA, and the top differentially expressed genes between VEH and LCMV were identical. This data suggests that methanol extraction does not influence RNA quality, integrity, or gene expression analysis. We observed that metRNA was associated with increased intra-replicate dispersion. This suggests that prior metabolite extraction may increase RNAseq variability and have deleterious effects on statistical power. Co-extraction of metabolites and RNA is viable for multi-omics of a single sample, but consideration of loss of statistical power due to increased variability should be considered.

Shared scientific resources, also known as core facilities, support a significant portion of the research conducted at biomolecular research institutions. The Association of Biomolecular Resource Facilities (ABRF) established the Committee on Core Rigor and Reproducibility (CCoRRe) to further its mission of integrating advanced technologies, education, and communication in the operations of shared scientific resources in support of reproducible research. In order to first assess the needs of the scientific shared resource community, the CCoRRe solicited feedback from ABRF members via a survey. The purpose of the survey was to gain information on how U.S. National Institutes of Health (NIH) initiatives on advancing scientific rigor and reproducibility influenced current services and new technology development. In addition, the survey aimed to identify the challenges and opportunities related to implementation of new reporting requirements and to identify new practices and resources needed to ensure rigorous research. The results revealed a surprising unfamiliarity with the NIH guidelines. Many of the perceived challenges to the effective implementation of best practices (i.e., those designed to ensure rigor and reproducibility) were similarly noted as a challenge to effective provision of support services in a core setting. Further, most cores routinely use best practices and offer services that support rigor and reproducibility. These services include access to well-maintained instrumentation and training on experimental design and data analysis as well as data management. Feedback from this survey will enable the ABRF to build better educational resources and share critical best-practice guidelines. These resources will become important tools to the core community and the researchers they serve to impact rigor and transparency across the range of science and technology.

Background The infectious cycle of varicella-zoster virus (VZV) after reactivation from the dorsal root ganglia includes replication and assembly of complete enveloped virions in the human skin to cause the characteristic herpes zoster (shingles). Methods To pursue studies of innate immunity to VZV infection, we have adapted a fetal skin organ culture model to a human neonatal foreskin explant model. Results Abundant expression of VZV IE62, gE, and gC was visualized by confocal microscopy while numerous enveloped virions were observed by electron microscopy in infected skin organ cultures. Microarray experiments demonstrated that the patterns of upregulated transcripts differed between VZV-infected cells and VZV-infected skin explants. One result stood out, namely a >30-fold elevated interleukin (IL)-6 level in the infected skin explant that was not present in the infected monolayer culture. The IL-6 results in the polyermase chain reaction (PCR) assay were reproduced by quantitative PCR testing with newly designed primers. To determine if increased transcription was accompanied by increased IL-6 expression, we quantitated the levels of IL-6 protein in the explant media at increasing intervals after infection. We found a statistically significant increase in IL-6 protein levels secreted into the media from VZV-infected skin explants as compared with mock-infected explants. Conclusions The cellular stress response to VZV infection in neonatal skin explants included highly elevated levels of IL-6 transcription and expression. This skin organ model could be adapted to other viruses with a skin tropism, such as herpes simplex virus.

Objective- Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. Approach and Results- Control, hypertensive, hypercholesterolemic (Apoe(-/-)), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. Conclusions- Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.