Project description:Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing . Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells.
Project description:Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions are recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) “Calling Cards” into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high performance computing environment and to conduct downstream analyses.
Project description:The ability to chronicle transcription factor binding events throughout the development of an organism would facilitate mapping of transcriptional networks that control cell fate decisions. We describe a method for permanently recording protein-DNA interactions in mammalian cells. We endow transcription factors with the ability to deposit a transposon into the genome near to where they bind. The transposon becomes a “Calling Card” the transcription factor leaves behind to record its visit to the genome. The locations of the Calling Cards can be determined by massively-parallel DNA sequencing. We show that the transcription factor SP1 fused to the piggyBac transposase directs insertion of the piggyBac transposon near SP1 binding sites. The locations of transposon insertions are highly reproducible, and agree with sites of SP1-binding determined by ChIP-seq. Genes bound by SP1 are more likely to be expressed in the HCT116 cell line we used, and SP1-bound CpG islands show a strong preference to be unmethylated. This method has the potential to trace transcription factor binding throughout cellular and organismal development in a way that has heretofore not been possible.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 16,015 nuclei in human adult testis. This dataset includes five samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.