Project description:A number of methods have been proposed to incorporate multiple scans at different intensities to reduce the quantification error and to minimize effects of saturation, but no direct comparison of their efficacy has been made. Here we used 40 technical replicates to compare individual scans at low, medium and high sensitivity with three methods for combining data from multiple scans (either 2 scan or 3 scan cases) using a variety of metrics Keywords: Multiple Scans
Project description:Post-hybridization washing is an essential part of microarray experiments. Both, the quality of the experimental washing protocol and the adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of washing cycles. Particularly, three Affymetrix GeneChip HGU133plus2 arrays were hybridized and equilibrated for 16 hours in the hybridization oven. For one of the three arrays washing and staining was performed according to the manufacturer’s instructions. For another array the first scan was done immediately after low stringent wash and staining without intermitting stringent washing. Then, the array was stringently washed and scanned in alternating order three more times where each washing step consists of a definite number of washing cycles. The third array was low stringently washed followed by two stringent washing cycles and staining before the first scan. Subsequently it was analogously processed as array A. All three chips are repeatedly processed in a second series of alternating wash/scan-cycles which was performed using the same protocol for each chip as in the first series as described above. As in the first series the arrays were also stained a second time to compensate for any loss of bleached fluorescent dye. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. The washing function allows calibrating probe intensities for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures especially in the limit of small and large values.
Project description:Proteomic experiments, particularly those addressing dynamic proteome properties, time series, or genetic diversity, require the analysis of large sample numbers. Despite significant advancements in proteomic technologies in recent years, further improvements are needed to accelerate measurement and enhance proteome coverage and quantitative performance. Previously, we demonstrated that incorporating a scanning MS2 dimension into data-independent acquisition methods (Scanning SWATH, or more generally scanning DIA) but also ion trapping, improves analytical depth and quantitative performance, especially in proteomic methods using fast chromatography. Here, we evaluate the scanning DIA approach combined with ion trapping via the Zeno trap in a method termed ZT Scan DIA, using a ZenoTOF 7600+ instrument (SCIEX). Applying this method to established proteome standards across various analytical setups, enabling intermediate to high sample throughput, we observed a 30–40% increase in identified precursors. This enhancement extended to overall protein identification and precise quantification. Furthermore, ZT Scan DIA effectively eliminated quantitative bias, as demonstrated by its ability to deconvolute proteomes in multi-species mixtures. We propose that ZT Scan DIA can be used to broaden the application in proteomics, particularly in studies requiring high quantitative precision with low sample input, post-translational modification (PTM) analysis, and high-throughput workflows.
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Micrococcol nuclease digestion and sequencing (MNase-seq) of human BJ fibroblasts
Project description:High-throughput mass spectrometry-based proteomics is gaining increasing interest, but mass spectrometers need to adapt to enable faster analysis times. For Orbitrap mass spectrometers, faster scan rates are constrained by limitations on ion accumulation time, duty cycle, and transient length. Here, we introduce a new scanning strategy called "pre-accumulation" that enables storing ions in the bent flatapole in parallel with the C-trap/IRM operation. This significantly improves ion beam utilization and allows scanning speeds over 70 Hz on hybrid Orbitrap instruments.The combination of pre-accumulation and increased scan speeds enhances peptide and protein group identifications for short LC gradients, and improves sensitivity for high-throughput applications. These benefits are further amplified when coupled with full mass range phase-constrained spectrum deconvolution (ΦSDM), especially for fast, lower-resolving Orbitrap measurements. The pre-accumulation approach offers distinct advantages, particularly for conditions with reduced signal input, and does not require any hardware changes, making it highly attractive for Orbitrap mass spectrometers used with fast MS/MS acquisition methods.
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Cleavage under Targets and Release Using Nuclease (CUT&RUN) of SOX2 and SOX2-DBD after ectopic expression in BJ fibroblasts.
Project description:Microarray was conducted on Illumina Human Ref-6 Version 2 Expression Chip (Illumina, San Diego, CA). Three independent cultures were used for RNA isolation with RNeasy plus mini kit (Qiagen, Valencia, CA). RNA quality was checked by Agilent Bioanalyzer (Agilent, Santa Clara, CA). An Ambion labeling kit was used for labeling cDNA followed by hybridization to Illumina chips. ChIP scan data was extracted by Illumina Beadstudio and subsequently analyzed using Bioconductor lumi package.
Project description:We developed SCAN-seq2, a high-throughput and highly sensitive single-cell RNA sequencing method based on the TGS platform. Our study demonstrated that SCAN-seq2 improves upon the previous method, SCAN-seq, in terms of sensitivity and throughput. By using reference-guided assembly of single-cell data, we were able to identify thousands of novel full-length RNA isoforms, including cell type-specific expression patterns of pseudogenes. We also accurately determined V(D)J rearrangement events in T and B cells. Lastly, we found that treatment of HepG2 and Hela cells with the spliceosome inhibitor Isoginkgetin (IGG) resulted in a subpopulation of cells with distinct apoptosis features. Our study provides a promising new tool for single-cell transcriptome research. The source code for SCAN-seq2 data analysis pipelines is available at https://github.com/liuzhenyu-yyy/SCAN-seq2 .
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for HNF4A, FOXA1, FOXA1-DBD, FOXA1-NHAA, FOXA1-RRAA, as well as H2B-HALO after ectopic expression in human BJ fibroblast cells.
Project description:In the rapidly moving proteomics field, a diverse patchwork of algorithms for data normalization and differential expression analysis is used by the community. We generated an all-inclusive mass spectrometry downstream analysis pipeline (MS-DAP) that integrates many algorithms for normalization and statistical analyses and produces standardized quality reporting with extensive data visualizations. Second, systematic evaluation of normalization and statistical algorithms on various benchmarking datasets, including additional data generated in this study, suggest best-practices for data analysis. Commonly used approaches for differential testing based on moderated t-statistics are consistently outperformed by more recent statistical models, all integrated in MS-DAP, and we encourage their adoption. Third, we introduced a novel normalization algorithm that rescues deficiencies observed in commonly used normalization methods. Finally, we used the MS-DAP platform to re-analyze a recently published large-scale proteomics dataset of CSF from AD patients. This revealed increased sensitivity, resulting in additional significant target proteins which improved overlap with results reported in related studies and includes a large set of new potential AD biomarkers in addition to previously reported.