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| Name | Authors | DOI | Group |
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Adult human small intestine cell dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.rnnd5de | Human Cell Atlas Method Development Community | Protocol for human small intestine cell dissociation, performed on ice to reduce artifact gene expression. | Cincinnati Children's Hospital Medical Center | 7 | 2018 | Andrew Potter 2018. Adult human small intestine cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rnnd5de | 2021-04-15 09:16:34 | |||
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Cell dissociation from nasal and bronchial brushings with cold-active protease for single-cell RNA-seq Resource Report Resource Website |
Laure-Emmanuelle Zaragosi, Pascal Barbry | DOI:10.17504/protocols.io.qubdwsn | Human Cell Atlas Method Development Community | This protocol provides details on the cell dissociation that should be performed to obtain single-cell suspensions from nasal epithelium brushings.Cell dissociation is performed at 4°C to avoid gene expression alterations and maximize viability. The typical cell number recovery is 200 000 - 300 000 for one brushing.Cell suspensions are suitable for single-cell RNA-sequencing protocols. | Universite Cote d’Azur, CNRS, IPMC, 06560 Valbonne, France, Universite Cote d’Azur, CNRS, IPMC, 06560 Valbonne, France | 2 | 2019 | Laure-Emmanuelle Zaragosi, Pascal Barbry 2019. Cell dissociation from nasal and bronchial brushings with cold-active protease for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.qubdwsn | 2021-04-15 09:17:00 | |||
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Nuclei Isolation for Single-Nuclei RNA sequencing Resource Report Resource Website |
Danh Truong, Salah-Eddine Lamhamedi-Cherradi, Joseph A. Ludwig | DOI:10.17504/protocols.io.bkacksaw | Human Cell Atlas Method Development Community, Ludwig Lab | We developed this protocol while trying to isolate single-nuclei from frozen liposarcoma tissue. We tested this protocol on a variety of PDX's. For fattier tissues, we use a modified protocol based on a different kit. Thanks to Dr. Luciano Martelotto for his assistance and tips on the first section. This protocol is based on prior work: ‘Frankenstein’ protocol, Nuclei Isolation Kit: Nuclei EZ Prep, and Minute™ Total Protein Extraction Kit for Adipose Tissues/Cultured Adipocytes. | MD Anderson Cancer Center, MD Anderson Cancer Center, MD Anderson Cancer Center | 1 | 2020 | Danh Truong, Salah-Eddine Lamhamedi-Cherradi, Joseph A. Ludwig 2020. Nuclei Isolation for Single-Nuclei RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bkacksaw | 2021-04-15 09:16:47 | |||
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Adult mouse liver dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.yd2fs8e | Human Cell Atlas Method Development Community | This protocol is used to dissociate adult (10 week) CD-1 mouse liver "on ice", preserving authentic gene expression profiles. The first layer consists of 3.5 hours rotation at 4 °C in 0.25% trypsin, and the second layer consists of 15 min. in bacillus licheniformis, with trituration on ice. The yield is 5000 cells/mg with 97% viability (as measured by trypan blue). | CCHMC | 2 | 2019 | Andrew Potter 2019. Adult mouse liver dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.yd2fs8e | 2021-04-15 09:16:46 | |||
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Thawing Cryopreserved Human Islets Resource Report Resource Website |
James Lyon, Patrick Macdonald, Jocelyn Manning Fox | DOI:10.17504/protocols.io.ui5eug6 | Human Cell Atlas Method Development Community | This protocol details the thawing of human islets following their cryopreservation, as performed by the Alberta Diabetes Institute IsletCore. The crypreservation method is detailed in Human Islet Cryopreservation protocol. Human islets cryopreserved and thawed using these methods have been found to retain viability and function after 20years of cryogenic biobanking.Manning Fox JE, Lyon J, Dai XQ, Wright RC, Hayward J, van de Bunt M, Kin T, Shapiro AMJ, McCarthy MI, Gloyn AL, Ungrin MD, Lakey JR, Kneteman NM, Warnock GL, Korbutt GS, Rajotte RV, MacDonald PE (2015) Human islet function following 20 years of cryogenic biobanking. Diabetologia, 58(7): 1503-1512. | University of Alberta, University of Alberta, University of Alberta | 1 | 2019 | James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Thawing Cryopreserved Human Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.ui5eug6 | 2021-04-15 09:16:49 | |||
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CITE-seq for PBMCs Resource Report Resource Website |
Nicola Wilson, Fernando Calero-Nieto, Javier Rodríguez-Ubreva, Bertie Gottgens, Esteban Ballestar, Roser Vento-Tormo | DOI:10.17504/protocols.io.bjhmkj46 | Human Cell Atlas Method Development Community, Coronavirus Method Development Community | This is a protocol for CITE-seq for PBMCs. It includes the steps for Thawing samples and Antibody Staining. | Cambridge Institute For Medical Research, United Kingdom, Cambridge Institute For Medical Research, United Kingdom, Epigenetics and ImmuneDisease Group, Josep Carreras Research Institute (IJC), 08916 Badalona,Barcelona, Spain, Cambridge Institute For Medical Research, United Kingdom, Epigenetics and ImmuneDisease Group, Josep Carreras Research Institute (IJC), 08916 Badalona,Barcelona, Spain, Wellcome Sanger Institute, Cambridge, UK | 1 | 2020 | Nicola Wilson, Fernando Calero-Nieto, Javier Rodríguez-Ubreva, Bertie Gottgens, Esteban Ballestar, Roser Vento-Tormo 2020. CITE-seq for PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhmkj46 | 2021-04-15 09:16:51 | |||
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Archival preservation of cell suspensions for scRNA-Seq Resource Report Resource Website |
Linas Mazutis, Ignas Masilionis | Human Cell Atlas Method Development Community, NCIHTAN | Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute, NY 10065, USA, Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute, NY 10065, USA | 1 | 2020 | Linas Mazutis, Ignas Masilionis 2020. Archival preservation of cell suspensions for scRNA-Seq . protocols.io https:// | 2021-04-15 09:17:02 | |||||
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Human Islet Cryopreservation Resource Report Resource Website |
James Lyon, Patrick Macdonald, Jocelyn Manning Fox | DOI:10.17504/protocols.io.spcediw | Human Cell Atlas Method Development Community | This protocol details the cryopreservation of human islets, as performed by the Alberta Diabetes Institute IsletCore. Human islets cryopreserved using this method have been found to retain viability and function after 20years of cryogenic biobanking.Manning Fox JE, Lyon J, Dai XQ, Wright RC, Hayward J, van de Bunt M, Kin T, Shapiro AMJ, McCarthy MI, Gloyn AL, Ungrin MD, Lakey JR, Kneteman NM, Warnock GL, Korbutt GS, Rajotte RV, MacDonald PE (2015) Human islet function following 20 years of cryogenic biobanking. Diabetologia, 58(7): 1503-1512. | University of Alberta, University of Alberta, University of Alberta | 1 | 2019 | James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Human Islet Cryopreservation. protocols.io https://dx.doi.org/10.17504/protocols.io.spcediw | 2021-04-15 09:16:47 | |||
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Intestine cell dissociation Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.nf3dbqn | Human Cell Atlas Method Development Community | Protocol for human intestine cell dissociation. | Cincinnati Children's Hospital Medical Center | 1 | 2018 | Andrew Potter 2018. Intestine cell dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nf3dbqn | 2021-04-15 09:17:03 | |||
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Breast tumours dissociation Resource Report Resource Website |
Samah El Ghamrasni | DOI:10.17504/protocols.io.7m9hk96 | Human Cell Atlas Method Development Community, CZI START Project | A protocol designed to dissociate fresh breast tissues (surgical specimens and biopsies) for single-cell RNAseq. The protocol has been demonstrated to work successfully with fresh and cryopreserved tissues. | Princess Margaret Cancer Research Centre | 1 | 2019 | Samah El Ghamrasni 2019. Breast tumours dissociation . protocols.io https://dx.doi.org/10.17504/protocols.io.7m9hk96 | 2021-04-15 09:17:02 | |||
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Adult mouse kidney dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.rd2d28e | Human Cell Atlas Method Development Community | Protocol for adult (8-10 week) mouse kidney dissociation performed on ice to reduce artifact gene expression. The final yield (post dead-cell removal) is 90K cells from 13 mg tissue with 91% viability, approximately 7,000 cells/mg tissue. Prior to dead cell removal the yield is 110K cells from 13 mg tissue with 81% viability. | Cincinnati Children's Hospital Medical Center | 4 | 2018 | Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rd2d28e | 2021-04-15 09:17:06 | |||
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Adult mouse lung cell dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.rird4d6 | Human Cell Atlas Method Development Community | This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability. | University of Cincinnati | 2 | 2018 | Andrew Potter 2018. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rird4d6 | 2021-04-15 09:14:23 | |||
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Single Cell and Single Nuclei Analysis Human Heart Tissue Resource Report Resource Website |
Monika Litvinukova, Eric Lindberg, Henrike Maatz, Hongbo Zhang, Michael Radke, Michael Gotthardt, Kourosh Saeb-Parsy, Sarah Teichmann, Norbert Hübner | DOI:10.17504/protocols.io.veae3ae | Human Cell Atlas Method Development Community | This protocol provides a walk through for complex processing of fresh human cardiac tissue followed by tissue dissociation for single cell RNA sequencing and nuclei isolation for single nuclei RNA sequencing. The protocol can be used for various regions of the heart muscle, including the ventricles, atria and septum. Each section looks slightly different and will contain different amount of tissue, meaning that the number of applications and the amount of stored tissue will vary.The protocol is also applicable to animal cardiac tissue with minor modifications adjusting for size and amount of the tissue. | Max Delbrück Center, Genetics and Genomics of Cardiovascular Diseases, Berlin, Germany, Max Delbrück Center, Genetics and Genomics of Cardiovascular Diseases, Berlin, Germany, Max Delbrück Center, Genetics and Genomics of Cardiovascular Diseases, Berlin, Germany, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom, Max Delbrück Centre, Neuromuscular and Cardiovascular Cell Biology, Berlin, Germany, Max Delbrück Centre, Neuromuscular and Cardiovascular Cell Biology, Berlin, Germany, Department of Surgery and Cambridge NIHR Biomedical Research Centre, Biomedical Campus, University of Cambridge, Cambridge, United Kingdom, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom; Theory of Condensed Matter Group, The Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom; European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, United Kingdom, Max Delbrück Center, Genetics and Genomics of Cardiovascular Diseases, Berlin, Germany; German Centre for Cardiovascular Research (DZHK), Berlin, Germany; Charité-Universitätsmedizin, Berlin, Germany; Berlin Institute of Health (BIH), Berlin, Germany | 1 | 2018 | Monika Litvinukova, Eric Lindberg, Henrike Maatz, Hongbo Zhang, Michael Radke, Michael Gotthardt, Kourosh Saeb-Parsy, Sarah Teichmann, Norbert Hübner 2018. Single Cell and Single Nuclei Analysis Human Heart Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.veae3ae | 2021-04-15 09:14:21 | |||
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Colonic epithelial cell isolation for Single Cell RNA-sequencing Resource Report Resource Website |
David Fawkner-Corbett | DOI:10.17504/protocols.io.ztrf6m6 | Human Cell Atlas Method Development Community | Protocol for isolation of single colonic epithelial cells from human endoscopic biopsies.Reference - https://doi.org/10.1038/s41586-019-0992-y | University of Oxford | 1 | 2019 | David Fawkner-Corbett 2019. Colonic epithelial cell isolation for Single Cell RNA-sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.ztrf6m6 | 2021-04-15 09:14:26 | |||
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mcSCRB-seq protocol Resource Report Resource Website |
Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard | DOI:10.17504/protocols.io.nrkdd4w | Human Cell Atlas Method Development Community | Single-cell RNA sequencing (scRNA-seq) has emerged as a central genome-wide method to characterize cellular identities and processes. Consequently, improving its sensitivity, flexibility and cost-efficiency can advance many research questions. Among the flexible plate-based methods, “Single-Cell RNA-Barcoding and Sequencing” (SCRB-seq) is one of the most sensitive and efficient ones. Here, we systematically evaluated experimental conditions of this protocol and find that adding polyethylene glycol considerably increases sensitivity by enhancing cDNA synthesis. Furthermore, using Terra polymerase increases efficiency due to a more even cDNA amplification that requires less sequencing of libraries. We combined these and other improvements to a new scRNA-seq library protocol we call “molecular crowding SCRB-seq” (mcSCRB-seq), which we show to be the most sensitive and one of the most efficient and flexible scRNA-seq methods to date. | Johannes W. Bagnoli, et al (2018) Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nature Communications 9:2937. doi: 10.1038/s41467-018-05347-6 | Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München, Ludwig-Maximilians-Universität München | https://www.nature.com/articles/s41467-018-05347-6 | 1 | 2018 | Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard 2018. mcSCRB-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.nrkdd4w | 2021-04-15 09:14:25 | |
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CGAP Human Spleen Dissociation, Tissue Stability Study Resource Report Resource Website |
Anna Wilbrey-Clark, Adam Hunter | DOI:10.17504/protocols.io.32rgqd6 | Human Cell Atlas Method Development Community | Mechanical dissociation of human spleen tissue to single cells for use in 10X chromium scRNA-Seq preparation. | Wellcome Trust Sanger Institute, HCA, CGaP | 1 | 2019 | Anna Wilbrey-Clark, Adam Hunter 2019. CGAP Human Spleen Dissociation, Tissue Stability Study. protocols.io https://dx.doi.org/10.17504/protocols.io.32rgqd6 | 2021-04-15 09:14:36 | |||
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sNucDrop-seq Protocol Resource Report Resource Website |
Peng Hu, Emily Fabyanic, Deborah Y. Kwon, Sheng Tang, Zhaolan Zhou, Hao Wu | DOI:10.17504/protocols.io.vfse3ne | Human Cell Atlas Method Development Community, Wu Lab @ UPenn | Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues such as adult mammalian brains is challenging. Here, we integrate sucrose-gradient-assisted purification of nuclei with droplet microfluidics to develop a highly scalable single-nucleus RNA-seq approach (sNucDrop-seq), which is free of enzymatic dissociation and nucleus sorting. By profiling ∼18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution,in vivo. | Hu P, Fabyanic E, Kwon DY, Tang S, Zhou Z, Wu H. Dissecting Cell-Type Composition and Activity-Dependent Transcriptional State in Mammalian Brains by Massively Parallel Single-Nucleus RNA-Seq. Mol Cell. 2017 Dec 7;68(5):1006-1015.e7. doi: 10.1016/j.molcel.2017.11.017. | Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA, Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA, Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA, Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA, Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA, Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA | https://www.cell.com/molecular-cell/fulltext/S1097-2765(17)30876-6?innerTabgraphical_S1097276517308766=#secsectitle0035 | 2 | 2018 | Peng Hu, Emily Fabyanic, Deborah Y. Kwon, Sheng Tang, Zhaolan Zhou, Hao Wu 2018. sNucDrop-seq Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.vfse3ne | 2021-04-15 09:14:30 | |
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osmFISH Resource Report Resource Website |
Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson | DOI:10.17504/protocols.io.psednbe | Human Cell Atlas Method Development Community, CZI Spatial Transcriptomics Protocol Repository, Neurodegeneration Method Development Community | osmFISH is a cyclic single molecule fluorescent in situ hybridization protocol used to quantify the expression level of specific transcripts in tissue sections by direct labeling of individual RNA molecules. The number of transcripts quantified in each round correspond the numbers of fluorophores available in the microscope setup. In order to quantify a large number of genes, osmFISH provides a method to remove the probes/labelling (stripping) from their targets and get the tissue ready for the next round of labeling - imaging - stripping.Even though the osmFISH protocol has been developed to perform multiple smFISH rounds, it can also be used as a quick and simple method for one-round smFISH. Furthermore, the protocol can be further extended to include more complex encoding/barcoding shemes that can be used to resolve a larger number of targets. | Karolinska Institute Stockholm, Karolinska Institute Stockholm, Technion - Israel Institute of Technology, Karolinska Institute Stockholm, Karolinska Institute Stockholm, Karolinska Institute Stockholm, Karolinska Institute Stockholm | http://linnarssonlab.org/osmFISH/ | 1 | 2018 | Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson 2018. osmFISH. protocols.io https://dx.doi.org/10.17504/protocols.io.psednbe | 2021-04-15 09:14:41 | ||
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Adult mouse lung cell dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.ymgfu3w | Human Cell Atlas Method Development Community | This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability. | CCHMC | 3 | 2019 | Andrew Potter 2019. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ymgfu3w | 2021-04-15 09:14:45 | |||
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Adult mouse kidney dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.q7adzie | Human Cell Atlas Method Development Community | Protocol for adult (8-10 week) mouse kidney dissociation, performed on ice to reduce artifact gene expression. | Cincinnati Children's Hospital Medical Center | 3 | 2018 | Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q7adzie | 2021-04-15 09:14:46 |
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