Methods in Molecular Biology 1979 Valentina Proserpio Editor Single Cell Methods Sequencing and Proteomics M ETHODS IN M OLECULAR B IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Single Cell Methods Sequencing and Proteomics Edited by Valentina Proserpio Department of Life Sciences and System Biology, University of Turin, Italian Institute for Genomic Medicine, IIGM Turin, Torino, Italy Editor Valentina Proserpio Department of Life Sciences and System Biology University of Turin, Italian Institute for Genomic Medicine, IIGM Turin Torino, Italy ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9239-3 ISBN 978-1-4939-9240-9 (eBook) https://doi.org/10.1007/978-1-4939-9240-9 © Springer Science+Business Media, LLC, part of Springer Nature 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Preface From the first mRNA-Seq whole-transcriptome analysis in 2009, in less than 10 years, many new technologies and strategies have been rapidly developed in order to analyze the genome, transcriptome, and proteome of individual cells, scaling from few to hundreds of thousands of cells analyzed at a time. Since then, many new biological questions have opened, and many laboratories across the world have utilized single-cell omics for their research, with a parallel massive increase in the number of publications regarding single cells. Keeping up with such a rapidly evolving technology is not an easy task, and for someone that enters the “single-cell field” for the first time, this might look like a maze, a jungle of choices and possibilities. The aim of this Methods in Molecular Biology (MIMB) book is to give readers a comprehensive overview of the available options for investigating biological questions at the level of individual cells and to help them in deciding which way is best to follow for different biological questions. Written by outstanding scientists in the field, the book is organized into eight parts that span from organizing a single-cell lab to performing single-cell DNA-Seq, RNA-Seq, and proteomic experiments. The book also covers single-cell epigenetics, single-cell multi-omics analysis, screening, and live imaging of individual cells. Each chapter lists all the materials required for the experiment and describes every protocol in a detailed, step-by-step manner, with all the precautions that should be taken when working with individual cells. The authors wrote every procedure for experts as well as for readers with no prior knowledge, making each experiment simple to perform in every lab equipped with the listed instrumentation. With very rich and detailed “Notes” sections, in which scientists included all the small tips and hints to best perform every protocol and to avoid common practical mistakes, I am confident that this book will represent a very powerful resource for any lab that will approach any experiment at the level of individual cells. I would like to thank Dr. Sarah Teichmann for introducing me to the “single-cell world,” Prof. John Walker for the opportunity to edit this book and for his constant guidance, and all the authors for their amazing job, their time, and their effort to make this book as perfect and as comprehensive as possible. Torino, Italy Valentina Proserpio v Acknowledgment Valentina Proserpio is supported by the Fondazione Umberto Veronesi. vii Contents Preface . ................................................................... v Contributors................................................................. xiii PART ILAB SETUP AND TISSUE PREPARATION 1 Setting Up a Single-Cell Genomic Laboratory.............................. 3 Lira Mamanova 2 Tissue Handling and Dissociation for Single-Cell RNA-Seq . ............... 9 Felipe A. Vieira Braga and Ricardo J. Miragaia PART II SINGLE CELL TRANCRIPTOMIC ANALYSIS 3 Full-Length Single-Cell RNA Sequencing with Smart-seq2 . ............... 25 Simone Picelli 4 CEL-Seq2—Single-Cell RNA Sequencing by Multiplexed Linear Amplification ........................................................... 45 Itai Yanai and Tamar Hashimshony 5 Single-Cell RNA-Seq by Multiple Annealing and Tailing-Based Quantitative Single-Cell RNA-Seq (MATQ-Seq) ........................... 57 Kuanwei Sheng and Chenghang Zong 6 Single-Cell RNA Sequencing with Drop-Seq . .............................. 73 Josephine Bageritz and Gianmarco Raddi 7 Chromium 10Â Single-Cell 30 mRNA Sequencing of Tumor-Infiltrating Lymphocytes. ..................................... 87 Marco De Simone, Grazisa Rossetti, and Massimiliano Pagani 8 Seq-Well: A Sample-Efficient, Portable Picowell Platform for Massively Parallel Single-Cell RNA Sequencing. ....................... 111 Toby P. Aicher, Shaina Carroll, Gianmarco Raddi, Todd Gierahn, Marc H. Wadsworth II, Travis K. Hughes, Chris Love, and Alex K. Shalek 9 Single-Cell Tagged Reverse Transcription (STRT-Seq) ...................... 133 Kedar Nath Natarajan 10 Single-Cell RNA-Sequencing of Peripheral Blood Mononuclear Cells with ddSEQ. .................................................... 155 Shaheen Khan and Kelly A. Kaihara 11 High-Throughput Single-Cell Real-Time Quantitative PCR Analysis. ........ 177 Liora Haim-Vilmovsky ix x Contents 12 Single-Cell Dosing and mRNA Sequencing of Suspension and Adherent Cells Using the PolarisTM System ............................ 185 Chad D. Sanada and Aik T. Ooi 13 Targeted TCR Amplification from Single-Cell cDNA Libraries ............... 197 Shuqiang Li and Kenneth J. Livak PART III SINGLE CELL GENOMIC AND EPIGENOMIC ANALYSIS 14 Sequencing the Genomes of Single Cells................................... 227 Veronica Gonzalez-Pena and Charles Gawad 15 Studying DNA Methylation in Single-Cell Format with scBS-seq ............. 235 Natalia Kunowska 16 Single-Cell 5fC Sequencing . ............................................. 251 Chenxu Zhu, Yun Gao, Jinying Peng, Fuchou Tang, and Chengqi Yi 17 ChIPmentation for Low-Input Profiling of In Vivo Protein–DNA Interactions . ............................................. 269 Natalia Kunowska and Xi Chen PART IV SINGLE CELL PROTEOMIC ANALYSIS 18 Immunophenotyping of Human Peripheral Blood Mononuclear Cells by Mass Cytometry . ............................................. 285 Susanne Heck, Cynthia Jane Bishop, and Richard Jonathan Ellis 19 Classification of the Immune Composition in the Tumor Infiltrate............ 305 Davide Brusa and Jean-Luc Balligand PART VSINGLE CELL MULTI OMIC ANALYSIS 20 Combined Genome and Transcriptome (G&T) Sequencing of Single Cells .......................................................... 319 Iraad F. Bronner and Stephan Lorenz 21 Simultaneous Profiling of mRNA Transcriptome and DNA Methylome from a Single Cell ............................................ 363 Youjin Hu, Qin An, Ying Guo, Jiawei Zhong, Shuxin Fan, Pinhong Rao, Xialin Liu, Yizhi Liu, and Guoping Fan 22 Simultaneous Targeted Detection of Proteins and RNAs in Single Cells .......................................................... 379 Aik T. Ooi and David W. Ruff PART VI SINGLE CELL SCREENING 23 CRISPR Screening in Single Cells . ..................................... 395 Johan Henriksson Contents xi PART VII SINGLE CELL LIVE IMAGING 24 Single-Cell Live Imaging . ............................................. 409 Toru Hiratsuka and Naoki Komatsu PART VIII SINGLE CELL DATA ANALYSIS 25 Differential Expression Analysis in Single-Cell Transcriptomics ............... 425 Luca Alessandrı`, Maddalena Arigoni, and Raffaele Calogero 26 A Bioinformatic Toolkit for Single-Cell mRNA Analysis ..................... 433 Kevin Baßler, Patrick Gu¨nther, Jonas Schulte-Schrepping, Matthias Becker, and Paweł Biernat Index . ................................................................... 457 Contributors TOBY P. A ICHER Ragon Institute of MGH, Harvard, and MIT, Cambridge, MA, USA; Department of Chemistry, Institute for Medical Engineering and Sciences (IMES), MIT, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA LUCA ALESSANDRI` Department of Molecular