Protocols   Select Another Resource Report Type

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for human small intestine cell dissociation, performed on ice to reduce artifact gene expression. 

Proper citation: Andrew Potter 2018. Adult human small intestine cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rnnd5de Copy   

•   Source:

Authors: Laure-Emmanuelle Zaragosi, Pascal Barbry
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

•   Source:

Authors: Danh Truong, Salah-Eddine Lamhamedi-Cherradi, Joseph A. Ludwig
Group: Human Cell Atlas Method Development Community, Ludwig Lab
Summary: 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.

Proper citation: 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 Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: 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). 

Proper citation: Andrew Potter 2019. Adult mouse liver dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.yd2fs8e Copy   

•   Source:

Authors: James Lyon, Patrick Macdonald, Jocelyn Manning Fox
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Thawing Cryopreserved Human Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.ui5eug6 Copy   

•   Source:

Authors: Nicola Wilson, Fernando Calero-Nieto, Javier Rodríguez-Ubreva, Bertie Gottgens, Esteban Ballestar, Roser Vento-Tormo
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: This is a protocol for CITE-seq for PBMCs. It includes the steps for Thawing samples and Antibody Staining.

Proper citation: 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 Copy   

•   Source:

Authors: Linas Mazutis, Ignas Masilionis
Group: Human Cell Atlas Method Development Community, NCIHTAN

Proper citation: Linas Mazutis, Ignas Masilionis 2020. Archival preservation of cell suspensions for scRNA-Seq . protocols.io https:// Copy   

•   Source:

Authors: James Lyon, Patrick Macdonald, Jocelyn Manning Fox
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Human Islet Cryopreservation. protocols.io https://dx.doi.org/10.17504/protocols.io.spcediw Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for human intestine cell dissociation. 

Proper citation: Andrew Potter 2018. Intestine cell dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nf3dbqn Copy   

•   Source:

Authors: Samah El Ghamrasni
Group: Human Cell Atlas Method Development Community, CZI START Project
Summary: 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.

Proper citation: Samah El Ghamrasni 2019. Breast tumours dissociation . protocols.io https://dx.doi.org/10.17504/protocols.io.7m9hk96 Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rd2d28e Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: 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. 

Proper citation: Andrew Potter 2018. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rird4d6 Copy   

•   Source:

Authors: Monika Litvinukova, Eric Lindberg, Henrike Maatz, Hongbo Zhang, Michael Radke, Michael Gotthardt, Kourosh Saeb-Parsy, Sarah Teichmann, Norbert Hübner
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

•   Source:

Authors: David Fawkner-Corbett
Group: Human Cell Atlas Method Development Community
Summary: Protocol for isolation of single colonic epithelial cells from human endoscopic biopsies.Reference - https://doi.org/10.1038/s41586-019-0992-y

Proper citation: David Fawkner-Corbett 2019. Colonic epithelial cell isolation for Single Cell RNA-sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.ztrf6m6 Copy   

•   Source:

Authors: Johannes Bagnoli, Christoph Ziegenhain, Aleksandar Janjic, Lucas Esteban Wange, Beate Vieth, Swati Parekh, Johanna Geuder, Ines Hellmann, Wolfgang Enard
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

•   Source:

Authors: Anna Wilbrey-Clark, Adam Hunter
Group: Human Cell Atlas Method Development Community
Summary: Mechanical dissociation of human spleen tissue to single cells for use in 10X chromium scRNA-Seq preparation. 

Proper citation: Anna Wilbrey-Clark, Adam Hunter 2019. CGAP Human Spleen Dissociation, Tissue Stability Study. protocols.io https://dx.doi.org/10.17504/protocols.io.32rgqd6 Copy   

•   Source:

Authors: Peng Hu, Emily Fabyanic, Deborah Y. Kwon, Sheng Tang, Zhaolan Zhou, Hao Wu
Group: Human Cell Atlas Method Development Community, Wu Lab @ UPenn
Summary: 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.

Proper citation: 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 Copy   

•   Source:

Authors: Simone Codeluppi, Lars Borm, amit zeisel, Gioele La Manno, Josina A. van Lunteren, Camilla I. Svensson, Sten Linnarsson
Group: Human Cell Atlas Method Development Community, CZI Spatial Transcriptomics Protocol Repository, Neurodegeneration Method Development Community
Summary: 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. 

Proper citation: 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 Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: 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. 

Proper citation: Andrew Potter 2019. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ymgfu3w Copy   

•   Source:

Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for adult (8-10 week) mouse kidney dissociation, performed on ice to reduce artifact gene expression.

Proper citation: Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q7adzie Copy   

•   Source: