CellTag Indexing: a genetic barcode-based multiplexing tool for single-cell technologies

Single-cell technologies have seen rapid advancements in recent years, along with new analytical challenges and opportunities. These high-throughput assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, ‘CellTag Indexing’, where we transduce and label samples that can then be pooled together for downstream application and analysis. By introducing predefined genetic barcodes that are transcribed and readily detected, we can reliably read out sample identity via genomic or transcriptomic profiling, permitting the simultaneous assessment of cell grouping and transcriptional state. We validate and demonstrate the utility of CellTag Indexing by sequencing transcriptomes at single-cell resolution using a variety of cell types including mouse pre-B cells, primary mouse embryonic fibroblasts, human HEK293T cells, and mouse induced endoderm progenitors. Furthermore, we establish CellTag Indexing as a valuable tool for multiplexing direct lineage reprogramming perturbation experiments. We present CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell RNA-sequencing and multiplexing strategies.

[1]  Samantha A. Morris,et al.  Dissecting Engineered Cell Types and Enhancing Cell Fate Conversion via CellNet , 2014, Cell.

[2]  Alexander van Oudenaarden,et al.  Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon , 2016, Proceedings of the National Academy of Sciences.

[3]  Eduard Batlle,et al.  Circulating IGF-I and IGFBP3 Levels Control Human Colonic Stem Cell Function and Are Disrupted in Diabetic Enteropathy. , 2015, Cell stem cell.

[4]  Samantha A. Morris,et al.  Single-cell analysis of clonal dynamics in direct lineage reprogramming: a combinatorial indexing method for lineage tracing , 2017, bioRxiv.

[5]  Daniel Wind,et al.  Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis. , 2006, Developmental biology.

[6]  Jonathan S. Weissman,et al.  MULTI-seq: Scalable sample multiplexing for single-cell RNA sequencing using lipid-tagged indices , 2018, bioRxiv.

[7]  Gunnar C. Hansson,et al.  The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host–microbial interactions , 2010, Proceedings of the National Academy of Sciences.

[8]  Andrew D. Rouillard,et al.  Enrichr: a comprehensive gene set enrichment analysis web server 2016 update , 2016, Nucleic Acids Res..

[9]  Bertrand Z. Yeung,et al.  Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics , 2018, Genome Biology.

[10]  Johan Ericson,et al.  The novel enterochromaffin marker Lmx1a regulates serotonin biosynthesis in enteroendocrine cell lineages downstream of Nkx2.2 , 2016, Development.

[11]  Avi Ma'ayan,et al.  Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool , 2013, BMC Bioinformatics.

[12]  Matthew E. Ritchie,et al.  limma powers differential expression analyses for RNA-sequencing and microarray studies , 2015, Nucleic acids research.

[13]  Lu Wen,et al.  Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing , 2018, Nature Cell Biology.

[14]  Grace X. Y. Zheng,et al.  Massively parallel digital transcriptional profiling of single cells , 2016, Nature Communications.

[15]  A. Germanà,et al.  Acid-sensing ion channel 2 (ASIC2) in the intestine of adult zebrafish , 2011, Neuroscience Letters.

[16]  Alexander van Oudenaarden,et al.  The Lgr 5 intestinal stem cell signature : robust expression of proposed quiescent ‘ þ 4 ’ cell markers , 2012 .

[17]  Xiangang Zou,et al.  Lrig1 marks a population of gastric epithelial cells capable of long-term tissue maintenance and growth in vitro , 2018, Scientific Reports.

[18]  Ying Liu,et al.  Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1 , 2009, Proceedings of the National Academy of Sciences.

[19]  Salah Ayoub,et al.  Cell fixation and preservation for droplet-based single-cell transcriptomics , 2017, BMC Biology.

[20]  Fiona M Watt,et al.  Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence , 2006, Proceedings of the National Academy of Sciences.

[21]  Atsushi Suzuki,et al.  Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming. , 2017, Cell stem cell.

[22]  Tony Pawson,et al.  Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Narinder Pal,et al.  Defining the transcriptomic landscape of the developing enteric nervous system and its cellular environment , 2017, BMC Genomics.

[24]  Jun Zhao,et al.  Removal of batch effects using distribution‐matching residual networks , 2016, Bioinform..

[25]  Michaela Frye,et al.  Lrig1 Expression Defines a Distinct Multipotent Stem Cell Population in Mammalian Epidermis , 2009, Cell stem cell.

[26]  R. Irizarry,et al.  Missing data and technical variability in single‐cell RNA‐sequencing experiments , 2018, Biostatistics.

[27]  S. Itzkovitz,et al.  Spatial Reconstruction of Single Enterocytes Uncovers Broad Zonation along the Intestinal Villus Axis , 2018, Cell.

[28]  Laleh Haghverdi,et al.  Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors , 2018, Nature Biotechnology.

[29]  Paul Hoffman,et al.  Integrating single-cell transcriptomic data across different conditions, technologies, and species , 2018, Nature Biotechnology.

[30]  Samantha A. Morris,et al.  Comparative Analysis and Refinement of Human PSC-Derived Kidney Organoid Differentiation with Single-Cell Transcriptomics. , 2018, Cell stem cell.

[31]  I. Amit,et al.  Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq , 2016, Cell.

[32]  Xiuli Wu,et al.  The Leucine-rich Repeat Protein LRIG1 Is a Negative Regulator of ErbB Family Receptor Tyrosine Kinases* , 2004, Journal of Biological Chemistry.

[33]  Lai Guan Ng,et al.  Dimensionality reduction for visualizing single-cell data using UMAP , 2018, Nature Biotechnology.

[34]  Samantha A. Morris,et al.  Single-cell mapping of lineage and identity in direct reprogramming , 2018, Nature.

[35]  Sjoerd Post,et al.  The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system , 2014, Immunological reviews.

[36]  Lori Sussel,et al.  Nkx2.2 regulates cell fate choice in the enteroendocrine cell lineages of the intestine. , 2008, Developmental biology.

[37]  B. Petersen,et al.  Insulin-like growth factor binding protein-3 is required for the regulation of rat oval cell proliferation and differentiation in the 2AAF/PHX model , 2010, Hepatic medicine : evidence and research.

[38]  Timothy K Lu,et al.  Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM , 2016, Proceedings of the National Academy of Sciences.

[39]  Andrew C. Adey,et al.  Single-Cell Transcriptional Profiling of a Multicellular Organism , 2017 .

[40]  Aki Masuda,et al.  Prox1 postmitotically defines dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus , 2012, Development.

[41]  Martin S. Taylor,et al.  The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Bruce J. Aronow,et al.  The Pan-ErbB Negative Regulator Lrig1 Is an Intestinal Stem Cell Marker that Functions as a Tumor Suppressor , 2012, Cell.

[43]  André F. Rendeiro,et al.  Pooled CRISPR screening with single-cell transcriptome read-out , 2017, Nature Methods.

[44]  Guillaume J. Filion,et al.  Starcode: sequence clustering based on all-pairs search , 2015, Bioinform..

[45]  Irving L. Weissman,et al.  Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding , 2011, Nature Biotechnology.

[46]  Sayaka Sekiya,et al.  Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors , 2011, Nature.

[47]  Dan Goldowitz,et al.  Scrambler and yotari disrupt the disabled gene and produce a reeler -like phenotype in mice , 1997, Nature.

[48]  Aviv Regev,et al.  Nuclei multiplexing with barcoded antibodies for single-nucleus genomics , 2018 .

[49]  H. Clevers,et al.  Identification of stem cells in small intestine and colon by marker gene Lgr5 , 2007, Nature.

[50]  Erik Sundström,et al.  RNA velocity of single cells , 2018, Nature.

[51]  Chun Jimmie Ye,et al.  Multiplexed droplet single-cell RNA-sequencing using natural genetic variation , 2017, Nature Biotechnology.

[52]  Esteban Ballestar,et al.  A robust and highly efficient immune cell reprogramming system. , 2009, Cell stem cell.

[53]  Duhee Bang,et al.  Multiplexed single-cell RNA-seq via transient barcoding for drug screening , 2018, bioRxiv.

[54]  Tetsuya Nakamura,et al.  Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon , 2014, Genes & development.

[55]  Hans Clevers,et al.  Lrig1 controls intestinal stem cell homeostasis by negative regulation of ErbB signalling , 2012, Nature Cell Biology.

[56]  Julia Holzmann,et al.  Prox1 identifies proliferating neuroblasts and nascent neurons during neurogenesis in sympathetic ganglia , 2015, Developmental neurobiology.

[57]  A. Acker-Palmer,et al.  Ephrin Bs are essential components of the Reelin pathway to regulate neuronal migration , 2011, Nature.

[58]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[59]  Berthold Göttgens,et al.  The Epidermis Comprises Autonomous Compartments Maintained by Distinct Stem Cell Populations , 2013, Cell stem cell.

[60]  Jens Hjerling-Leffler,et al.  Transcription and Signaling Regulators in Developing Neuronal Subtypes of Mouse and Human Enteric Nervous System , 2017, Gastroenterology.

[61]  Thomas M. Norman,et al.  Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens , 2016, Cell.

[62]  Thomas M. Norman,et al.  A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response , 2016, Cell.

[63]  S. Orkin,et al.  Mapping the Mouse Cell Atlas by Microwell-Seq , 2018, Cell.

[64]  Lior Pachter,et al.  Highly Multiplexed Single-Cell RNA-seq for Defining Cell Population and Transcriptional Spaces , 2018, bioRxiv.