pCAG- WIPI2dI92E-cs- TEV -STREP
By onPlasmid: Mammalian expression of human WIPI2d I92E with C-terminal Strep.
VPS13D DNA plasmid generation
By onThis protocol describes the basic mole cular cloning technique utilized for the generation of VPS13D constructs in https://doi.org/10.1083/jcb.202010004. This protocol and the enzymes included in it are commercialized by Takara Bio.Due to low expression of big DNA constructs (u003e10kb), we decided to generate a codon-optimized cDNA encoding for human VPS13D, including an mScarlet fluorescent protein after aminoacid 1576 flanked by BamHI restriction enzyme sites. The construct was generated by and purchased from Genscript. From this initial construct, we generated several VPS13D constructs. The specific primers and enzymes used for each construct are included in Table 1 of our manuscript: https://doi.org/10.1083/jcb.202010004" . For most of our cloning procedures, Infusion cloning (Takara) was used.
From Policy to Practice: Tracking an Open Science Funding Initiative
By onBy normalizing the open science and compliance process across funding bodies, ASAP hopes to simplify and streamline researcher, institutional, and funder workflows, allowing researchers to focus on science.
Detecting Full-Length EccDNA with FLED and long-reads sequencing
By onReconstructing the full-length sequence of extrachromosomal circular DNA (eccDNA) from short sequencing reads has proved challenging given the similarity of eccDNAs and their corresponding linear DNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length eccDNAs. Here we describe a new strategy that combined rolling circle amplification (RCA) and nanopore long-reads sequencing technology to generate full-length eccDNAs. We further developed a novel algorithm, called Full-Length eccDNA Detection (FLED), to reconstruct the sequence of eccDNAs. We used FLED to analyze seven human epithelial and cancer cell line samples and identified over 5,000 full-length eccDNAs per sample. The structures of identified eccDNAs were validated by both PCR and Sanger sequencing. Compared to other published nanopore-based eccDNA detectors, FLED exhibited higher sensitivity. In cancer cell lines, the genes overlapped with eccDNA regions were enriched in cancer-related pathways and cis-regulatory elements can be predicted in the up-stream or downstream of intact genes on eccDNA molecules, and the expressions of these cancer-related genes were dysregulated in tumor cell lines, indicating the regulatory potency of eccDNAs in biological processes. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length eccDNA sequences. FLED is imple-mented using Python3 which is freely available on GitHub (https://github.com/FuyuLi/FLED).
Generation of human-induced pluripotent-stem-cell-derived cortical neurons for high-throughput imaging of neurite morphology and neuron maturation
By onA STAR protocol that describes how to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis
Phosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.
By onPhosphosite mapping on PI3Kc1 by ULK1 and TBK1 kinases.
Conserved and cell type-specific transcriptional responses to IFN-γ in the ventral midbrain
By onNeurons present antigens when exposed to IFN-γ, leading to MHC-I expression. IFN-γ induces MHC-I in neurons, glia, and microglia, with glia showing a stronger response. Neuronal response to IFN-γ is dependent on IFNGR signaling.
Purifying Vps13-VAB domain proteins
By onIn this protocol, we have listed the steps to purify the Vps13 adaptor binding domain (or VAB) with or without PxP motif fusion peptide derived from adaptor proteins Mcp1, Ypt35 or Spo71.
Detection of mosaic and population-level structural variants with Sniffles2
By onSniffles2 is a fast and accurate tool for identifying complex genomic alterations using long -read data.
Native-PAGE analysis of VCP hexamer
By onThis protocol is associated with the following preprint, published on February 19th 2022: The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
Single cell transcriptomics from DAT-Cre mouse gut tissue
By onColonic myenteric cells were dissociated, and live tdTomato-positive cells were FACS-collected. Colons were isolated from 2 male and female wild type adult DAT-Cre mice. Wild type C57Bl6/J was used as control for gating. Cells were processed according to 10X Genomics Chromium single cell 3' Reagent guidelines.
Cell lysis and gel electrophoresis for protein analysis of HeLa cells
By onThe authors present multiple protocols used for biochemical analysis of protein expression and association. T
Immunoblotting analysis of samples from GolgiTAG (TMEM115-3HA) immunoprecipitation
By onAn immunoblotting method to assess efficient enrichment of Golgi proteins in Golgi immunoprecipitation products compared to whole cell lysates, and the purity of the immunoprecipitated Golgi by monitoring the expression of other organelles’ markers.
Sectioning of Mouse Brain by Cryostat
By onThis protocol describes how to use the cryostat to prepare and slice mouse brain sections for Immunohistochemistry.
Therapeutic Potential of PTB Inhibition Through Converting Glial Cells to Neurons in the Brain
By onCell replacement therapy represents a promising approach for treating neurodegenerative diseases. Contrary to the common addition strategy to generate new neurons from glia by overexpressing a lineage-specific transcription factor(s), a recent study introduced a subtraction strategy by depleting a single RNA-binding protein, Ptbp1, to convert astroglia to neurons not only in vitro but also in the brain. Given its simplicity, multiple groups have attempted to validate and extend this attractive approach but have met with difficulty in lineage tracing newly induced neurons from mature astrocytes, raising the possibility of neuronal leakage as an alternative explanation for apparent astrocyte-to-neuron conversion. This review focuses on the debate over this critical issue. Importantly, multiple lines of evidence suggest that Ptbp1 depletion can convert a selective subpopulation of glial cells into neurons and, via this and other mechanisms, reverse deficits in a Parkinson's disease model, emphasizing the importance of future efforts in exploring this therapeutic strategy.
