Single-cell isolation

Y. Hu, S.G. Tattikota, Y. Liu, A. Comjean, Y. Gao, C. Forman, G. Kim, J. Rodiger, I. Papatheodorou, G. dos Santos, S.E. Mohr, and N. Perrimon. Submitted. “DRscDB: A single-cell RNA-seq resource for data mining and data comparison across species.” BioRxiv. Publisher's VersionAbstract
With the advent of single-cell RNA sequencing (scRNA-seq) technologies, there has been a spike in studies involving scRNA-seq of several tissues across diverse species including Drosophila. Although a few databases exist for users to query genes of interest within the scRNA-seq studies, search tools that enable users to find orthologous genes and their cell type-specific expression patterns across species are limited. Here, we built a new search database, called DRscDB ( to address this need. DRscDB serves as a comprehensive repository for published scRNA-seq datasets for Drosophila and the relevant datasets from human and other model organisms. DRscDB is based on manual curation of Drosophila scRNA-seq studies of various tissue types and their corresponding analogous tissues in vertebrates including zebrafish, mouse, and human. Of note, our search database provides most of the literature-derived marker genes, thus preserving the original analysis of the published scRNA-seq datasets. DRscDB serves as a web-based user interface that allows users to mine, utilize and compare gene expression data pertaining to scRNA-seq datasets from the published literature.
A.M. Conard, N. Goodman, Hu, Y, N. Perrimon, R. Singh, C. Lawrence, and E. Larschan. Submitted. “TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data.” BioRxiv. Publisher's VersionAbstract
Uncovering how transcription factors (TFs) regulate their targets at the DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) in normal and diseased states. RNA-seq has become a standard method to measure gene regulation using an established set of analysis steps. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods which integrate ordered RNA-seq data with transcription factor binding data are urgently needed. Here, we present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to predict causal regulatory mechanism networks between TFs from time series multi-omics data. We used TIMEOR to identify a new link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at
Justin A Bosch, Shannon Knight, Oguz Kanca, Jonathan Zirin, Donghui Yang-Zhou, Yanhui Hu, Jonathan Rodiger, Gabriel Amador, Hugo J Bellen, Norbert Perrimon, and Stephanie E Mohr. 2020. “Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.” Curr Protoc Mol Biol, 130, 1, Pp. e112.Abstract
The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.
Benjamin E Housden, Shuailiang Lin, and Norbert Perrimon. 2014. “Cas9-based genome editing in Drosophila.” Methods Enzymol, 546, Pp. 415-39.Abstract

Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.

2014_Methods Enzymol_Housden.pdf