Biology subjects

Boenisch, U.

Publications and source records attributed to Boenisch, U..

5 recordsLinked to original sources

bioRxiv2024

Enhancing single-cell ATAC sequencing with formaldehyde fixation, cryopreservation, and multiplexing for flexible analysis

The assay for transposase-accessible chromatin using sequencing (ATAC-seq) revolutionized the field of epigenetics since its emergence by providing a means to uncover chromatin dynamics and other factors affecting gene expression. The development of single-cell (sc) applications in recent years led to an even deeper understanding of cell type specific gene regulatory mechanisms. One of the major challenges while running ATAC-seq experiments, bulk or sc, is the need for freshly collected cells for successful experiments. While various freezing methods have already been tested and established for bulk and sc ATAC-seq, quality metrics for preserved cells are rather poor or dependent on sampling time when compared to fresh samples. This makes it difficult to conduct all sorts of complex experiments i.e. with multiple conditions, patients, or time course studies. Especially, accounting for batch effects can be difficult if samples need to be processed at different time points of collection. We tackled this issue by adding a fixation step prior to the freezing method. The additional fixation step improved library quality and yield data comparable to fresh samples. The workflow was also tested on multiplexed sc ATAC experiments, set-up for cost-efficient low input sample handling. Sample cross-in, typically encountered in Tn5-based multiplex approaches, were tackled with a computational procedure specifically developed for this approach.

genomics↗

bioRxiv2020

AutoRELACS: Automated Generation And Analysis Of Ultra-parallel ChIP-seq

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method used to profile protein-DNA interactions genome-wide. RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ) is a recently developed ChIP-seq protocol that deploys a chromatin barcoding strategy to enable standardized and high-throughput generation of ChIP-seq data. The manual implementation of RELACS is constrained by human processivity in both data generation and data analysis. To overcome these limitations, we have developed AutoRELACS, an automated implementation of the RELACS protocol using the liquid handler Biomek i7 workstation. We match the unprecedented processivity in data generation allowed by AutoRELACS with the automated computation pipelines offered by snakePipes. In doing so, we build a continuous workflow that streamlines epigenetic profiling, from sample collection to biological interpretation. Here, we show that AutoRELACS successfully automates chromatin barcode integration, and is able to generate high-quality ChIP-seq data comparable with the standards of the manual protocol, also for limited amounts of biological samples.

genomics↗

bioRxiv2018

Parkour LIMS: facilitating high-quality sample preparation in next generation sequencing

SummaryThis paper presents Parkour, a software package for sample processing and quality management of next generation sequencing data and samples. Starting with user requests, Parkour allows tracking and assessing samples based on predefined quality criteria through different stages of the sample preparation workflow. Ideally suited for academic core laboratories, the software aims to maximize efficiency and reduce turnaround time by intelligent sample grouping and a clear assignment of staff to work units. Tools for automated invoicing, interactive statistics on facility usage and simple report generation minimize administrative tasks. Provided as a web application, Parkour is a convenient tool for both deep sequencing service users and laboratory personal. A set of web APIs allow coordinated information sharing with local and remote bioinformaticians. The flexible structure allows workflow customization and simple addition of new features as well as the expansion to other domains.\n\nAvailability and implementationThe code and documentation are available at https://github.com/maxplanck-ie/parkour\n\nContactboenisch@ie-freiburg.mpg.de

genomics↗

bioRxiv2018

Ultra-parallel ChIP-seq by barcoding of intact nuclei

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is an invaluable tool for mapping chromatin-associated proteins. However, sample preparation is still a largely individual and labor-intensive process that hinders assay throughput and comparability. Here, we present a novel method for ultra-parallelized high-throughput ChIP-seq that addresses the aforementioned problems. The method, called RELACS (Restriction Enzyme-based Labeling of Chromatin in Situ), employs barcoding of chromatin within intact nuclei extracted from different sources (e.g. tissues, treatments, time points). Barcoded nuclei are pooled and processed within the same ChIP, for maximal comparability and significant workload reduction. The choice of user-friendly, straightforward, enzymatic steps for chromatin fragmentation and barcoding makes RELACS particularly suitable for implementation large-scale clinical studies and scarce samples. RELACS can generate ChIP-seq libraries from hundreds of samples within three days and with less than 1000 cells per sample.

genomics↗

bioRxiv2017

The Polycomb-dependent epigenome controls β-cell dysfunction, dedifferentiation and diabetes

Chromatin is the physical template that stabilizes and specifies transcriptional programs. To date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combined deep epigenome mapping with single cell transcriptomics to mine for evidence of chromatin dysregulation in type-2 diabetes. We identify two chromatin-state signatures that track the trajectory of {beta}-cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-domains and a loss of expression at a subclass of highly active domains containing key lineage-defining genes. {beta}-cell specific deletion of Polycomb (Eed/PRC2) triggers parallel transcriptional signatures. Intriguingly, these {beta}-cell Eed-knockouts also exhibit highly penetrant hyperglycemia-independent dedifferentiation indicating that Polycomb dysregulation sensitizes the {beta}-cell for dedifferentiation. These findings provide novel resources for exploring transcriptional and epigenetic control of {beta}-cell (dys)function. They identify PRC2 as necessary for long-term maintenance of {beta}-cell identity. The data suggest a two-hit model for loss of {beta}-cell identity in diabetes and highlight epigenetic therapeutic potential to block dedifferentiation.

genomics↗