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| Name | Authors | DOI | Group |
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Associated Publications |
RRIDs used | ||||||
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Purification of PBMCs Resource Report Resource Website |
Roser Vento-Tormo | DOI:10.17504/protocols.io.bjinkkde | Human Cell Atlas Method Development Community, Coronavirus Method Development Community | This protocol details the steps and methods of PBMC purification | Wellcome Sanger Institute, Cambridge, UK | 1 | 2020 | Roser Vento-Tormo 2020. Purification of PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.bjinkkde | 2021-04-15 09:14:49 | |||
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Dissociation of Single Cell Suspensions from Human Breast Tissues Resource Report Resource Website |
Shanshan Bai, Emi Sei, Nicholas E. Navin | DOI:10.17504/protocols.io.t3geqjw | Human Cell Atlas Method Development Community | This protocol was developed for the human breast cell atlas (HBCA) project to obtain high-viability cell suspensions from freshly dissociated breast tissues from human patients. There are two options for performing this protocol: rapid-dissociation (15-30 min) or exhaustive dissociation (overnight). In our experience the rapid dissociation protocol will provide higher cell viability and better representation of breast cell types, however the total number of cells dissociated may be lower after enumeration. In contrast the exhaustive protocol provides higher cell numbers and maintains good cell viability but may result in skewing of cell type representations over the long time period. The rapid-dissociation protocol was used to generate most of the cell suspensions for the MD Anderson human breast cell atlas project and has been optimized and tested more extensively. | University of Texas MD Anderson Cancer Center, University of Texas MD Anderson Cancer Center, University of Texas MD Anderson Cancer Center | 1 | 2018 | Shanshan Bai, Emi Sei, Nicholas E. Navin 2018. Dissociation of Single Cell Suspensions from Human Breast Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.t3geqjw | 2021-04-15 09:14:54 | |||
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Static Glucose-stimulated Insulin Secretion (GSIS) Protocol - Human Islets Resource Report Resource Website |
Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald | DOI:10.17504/protocols.io.wztff6n | Human Cell Atlas Method Development Community, CIRTNR2FIC | University of Alberta, University of Alberta, University of Alberta | 3 | 2019 | Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald 2019. Static Glucose-stimulated Insulin Secretion (GSIS) Protocol - Human Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.wztff6n | 2021-04-15 09:14:52 | ||||
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AutoCUT&RUN: genome-wide profiling of chromatin proteins in a 96 well format on a Biomek Resource Report Resource Website |
Derek Janssens, Kami Ahmad, Steven Henikoff | DOI:10.17504/protocols.io.ufeetje | Human Cell Atlas Method Development Community | CUT&RUN is an antibody-targeted nuclease-cleavage method that profiles the genome-wide occupancy of DNA-binding proteins, histones and chromatin modifying proteins in situ with exceptional sensitivity and resolution. Here we provide a protocol to perform CUT&RUN in an automated 96 well format using a Beckman Biomek FX liquid handling robot equipped for magnetic separation and temperature control. To circumvent the need to purify small amounts of DNA prior to library preparation, we developed a method to polish the DNA ends in chromatin fragments for direct ligation of Illumina library adapters. This allows the samples to be kept in a 96 well format throughout the DNA end-polishing and adapter ligation steps. Deproteinated CUT&RUN libraries are purified on the Biomek using Ampure XP magnetic beads both before and after PCR enrichment. This AutoCUT&RUN protocol allows a single operator to generate up to 96 libraries in two days that are ready to be pooled and sequenced. The easy, cost-effective workflow makes AutoCUT&RUN an attractive tool for high-throughput characterization of chromatin features, and because AutoCUT&RUN generates high-quality profiles from frozen tissue samples this method is also suitable for rapidly profiling the epigenetic content of patient samples. | Janssens DH, Wu SJ, Sarthy JF, Meers MP, Myers CH, Olson JM, Ahmad K, Henikoff S, Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs. Epigenetics & Chromatin doi: 10.1186/s13072-018-0243-8 | Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center | https://doi.org/10.1186/s13072-018-0243-8 | 1 | 2018 | Derek Janssens, Kami Ahmad, Steven Henikoff 2018. AutoCUT&RUN: genome-wide profiling of chromatin proteins in a 96 well format on a Biomek. protocols.io https://dx.doi.org/10.17504/protocols.io.ufeetje | 2021-04-15 09:14:40 | |
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ADI IsletCore Protocols for the Isolation, Assessment and Cryopreservation of Human Pancreatic Islets of Langerhans for Research Purposes Resource Report Resource Website |
James Lyon, Aliya Spigelman, Patrick Macdonald, Jocelyn Manning Fox | DOI:10.17504/protocols.io.x3mfqk6 | Human Cell Atlas Method Development Community, CIRTNR2FIC | This collection of protocols describe the processes used by the Alberta Diabetes Institute IsletCore for the isolation of human pancreatic islets of Langerhans for research. In addition, protocols related to quality control and the cryopreservation of human islets are included.The ADI IsletCore program is described in full here:Lyon J, Manning Fox JE, Spigelman AF, Kim R, Smith N, O'Gorman D, Kin T, Shapiro AMJ, Rajotte RV, MacDonald PE (2016) Research-focused isolation of human islets from donors with and without diabetes at the Alberta Diabetes Institute IsletCore. Endocrinology, 157(2): 560-569. | University of Alberta, University of Alberta, University of Alberta, University of Alberta | 1 | 2019 | James Lyon, Aliya Spigelman, Patrick Macdonald, Jocelyn Manning Fox 2019. ADI IsletCore Protocols for the Isolation, Assessment and Cryopreservation of Human Pancreatic Islets of Langerhans for Research Purposes. protocols.io https://dx.doi.org/10.17504/protocols.io.x3mfqk6 | 2021-04-15 09:14:40 | |||
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Nuclei isolation from mouse lung for single nucleus RNASeq Resource Report Resource Website |
Jeffrey Koenitzer, Ben Humphreys | DOI:10.17504/protocols.io.bdv2i68e | Human Cell Atlas Method Development Community | This protocol is for nuclei isolation from mouse lung for single nucleus RNASeq.It is adapted directly from Joshi et al., with adjustments to RNase inhibitor concentrations and removal of FACS sorting steps. | Division of Pulmonary and Critical Care, Department of Medicine, Washington University in St. Louis School of Medicine, USA, Division of Nephrology, Department of Medicine, Washington University in St. Louis School of Medicine, USA; Department of Developmental Biology, Washington University in St. Louis School of Medicine, USA | 1 | 2020 | Jeffrey Koenitzer, Ben Humphreys 2020. Nuclei isolation from mouse lung for single nucleus RNASeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bdv2i68e | 2021-04-15 09:14:42 | |||
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Human Heart Nuclei Isolation for DroNc-seq Resource Report Resource Website |
Stephanie Lozano | DOI:10.17504/protocols.io.vgue3ww | Human Cell Atlas Method Development Community | Recent advances in single cell mRNA sequencing technology such as Drop-seq (Cell, 2015) have allowed the investigation of cellular composition and processes within complex tissues. Although some fresh tissue samples are available for prospective studies, there is an abundance of archived clinical samples readily available in biobanks to investigate different biological processes. However, archived tissues are not easily amenable to tissue digestion, yielding cells with compromised integrity. An alternate method to single-cell sequencing called DroNc-seq (Nature Methods, 2017) employs the use of single nuclei. DroNc-seq takes advantage of the higher structural integrity of the nuclear membrane as compared to the cellular membrane. This permits exploration of tissues for which a viable single-cell suspension cannot be obtained, such as cardiac tissue. With growing interest in cataloguing cardiac transcriptomic data for the Human Cell Atlas Project, the need for optimized nuclei isolation protocols proves critical. Here, we develop a method for obtaining single-nucleus suspensions from snap-frozen human heart tissue for use in DroNc-seq, expanding the variety of samples from which we can obtain transcriptomic data. | Basu Lab, University of Chicago | 2 | 2018 | Stephanie Lozano 2018. Human Heart Nuclei Isolation for DroNc-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.vgue3ww | 2021-04-15 09:15:03 | |||
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IsletCore Equipment Setup for Human Islet Isolation Resource Report Resource Website |
Patrick Macdonald, James Lyon, Jocelyn Manning Fox | DOI:10.17504/protocols.io.sg4ebyw | Human Cell Atlas Method Development Community | University of Alberta, University of Alberta, University of Alberta | 1 | 2019 | Patrick Macdonald, James Lyon, Jocelyn Manning Fox 2019. IsletCore Equipment Setup for Human Islet Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.sg4ebyw | 2021-04-15 09:14:59 | ||||
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Thymus dissociation Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.ngcdbsw | Human Cell Atlas Method Development Community | Protocol for thymus dissociation (10-week old CD-1 female). | Cincinnati Children's Hospital Medical Center | 1 | 2018 | Andrew Potter 2018. Thymus dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.ngcdbsw | 2021-04-15 09:14:45 | |||
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Drop-Seq Laboratory Protocol Resource Report Resource Website |
Evan Macosko, Melissa Goldman | DOI:10.17504/protocols.io.mkbc4sn | Human Cell Atlas Method Development Community | We have continued to optimize this protocol since doing the experiments in the Macosko et al. Cell paper. What we are sharing here is our current, optimized protocol. As a result, this protocol will not precisely match the methods section of the paper, which is a description of the experiments done in the paper. Please feel free to use these optimizations without author consideration - just acknowledge “helpful advice from Evan Macosko, Melissa Goldman and Steve McCarroll”, and please also include the URL and version number of the protocol in your methods section. This protocol also includes hints, suggestions and images that we could not fit into the the methods section of the paper. | Steve McCarroll’s lab, Harvard Medical School, Steve McCarroll’s lab, Harvard Medical School | http://mccarrolllab.com/dropseq/ | 1 | 2018 | Evan Macosko, Melissa Goldman 2018. Drop-Seq Laboratory Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mkbc4sn | 2021-04-15 09:14:50 | ||
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Single-cell RNA-Seq expression analysis Resource Report Resource Website |
John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev | DOI:10.17504/protocols.io.pbidike | Human Cell Atlas Method Development Community | This is a Single-cell RNA-Seq expression analysis Support Protocol for Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. | Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA, Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338574/#R8 | 1 | 2018 | John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev 2018. Single-cell RNA-Seq expression analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.pbidike | 2021-04-15 09:15:11 | ||
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Human Spinal Cord Nuclei Isolation Resource Report Resource Website |
Abbas Rizvi, Elena Kandror, Tom Maniatis | DOI:10.17504/protocols.io.9yhh7t6 | Human Cell Atlas Method Development Community | [Zuckerman Mind Brain Behavior Insitute, Columbia University], [Zuckerman Mind Brain Behavior Insitute, Columbia University], [Zuckerman Mind Brain Behavior Insitute, Columbia University] | 1 | 2019 | Abbas Rizvi, Elena Kandror, Tom Maniatis 2019. Human Spinal Cord Nuclei Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.9yhh7t6 | 2021-04-15 09:14:51 | ||||
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scNMT-seq Resource Report Resource Website |
Stephen Clark | DOI:10.17504/protocols.io.3b8girw | Human Cell Atlas Method Development Community | Here we describe the full wetlab protocol for scNMT-seq (single-cell nucleosome position, methylome and transcriptome sequencing), a plate-based single-cell multi-omic method.In scNMT-seq, a GpC methyltransferase is used to label non-nucleosome bound DNA thus encoding chromatin accessiblity information within the genomic DNA. Bisulfite conversion is then used to discriminate the two chromatin states which can be read from GpC dinucleotide positions. Endogenous DNA methylation is read from the CpG positions of the same molecules and gene expression data is obtained by capturing poly-A RNA on magnetic beads and pysically separating before the gDNA is bisulfite converted. This mRNA on beads is pocessed using Smart-seq2. The protocol is carried out in 96w plates and typically takes 3-4 days to complete. | Babraham Institute, Cambridge | 2 | 2019 | Stephen Clark 2019. scNMT-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.3b8girw | 2021-04-15 09:14:51 | |||
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P1 Kidney Cold-Active Protease Single Cell Dissociation Resource Report Resource Website |
Andrew Potter, Steve Potter | DOI:10.17504/protocols.io.nzkdf4w | Human Cell Atlas Method Development Community | Method used to derive single cell suspension from P1 mouse kidneys on ice, generating a cell suspension with greatly reduced artifact gene expresion changes and suitable for downstream analysis using 10x Chromium or DropSeq. | CCHMC, CCHMC | 1 | 2018 | Andrew Potter, Steve Potter 2018. P1 Kidney Cold-Active Protease Single Cell Dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.nzkdf4w | 2021-04-15 09:14:51 | |||
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CITE-seq Resource Report Resource Website |
Marlon Stoeckius | DOI:10.17504/protocols.io.ngydbxw | Human Cell Atlas Method Development Community | This protocol is for performing CITE-seq only. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) is a multimodal single cell phenotyping method developed in the Technology Innovation lab at the New York Genome Center in collaboration with the Satija lab.CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout. Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. 10x Genomics, Drop-seq, ddSeq).This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches. | New York Genome Center Technology Innovation Lab | https://cite-seq.com/ | 1 | 2018 | Marlon Stoeckius 2018. CITE-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.ngydbxw | 2021-04-15 09:15:17 | ||
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RNA Imaging with MERFISH - Sample Preparation and Staining Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.merc3d6 | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | The preparation and staining of samples for MERFISH follows closely the typical protocols used for smFISH (Raj et al., 2008). However, there are a few places in which we have modified these protocols to optimize MERFISH staining. Again, RNase contamination can destroy samples, so care should be taken to work in an RNase-free environment. | Harvard University, Cambridge, Massachusetts, 02138 USA, Harvard University, Cambridge, Massachusetts, 02138 USA | https://www.ncbi.nlm.nih.gov/pubmed/27241748 | 1 | 2018 | Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Sample Preparation and Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.merc3d6 | 2021-04-15 09:15:18 | ||
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Frozen tissue dissociation for single-nucleus RNA-Seq Resource Report Resource Website |
Linas Mazutis, Ignas Masilionis, Ojasvi Chaudhary | Human Cell Atlas Method Development Community, NCIHTAN | Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center | 1 | 2020 | Linas Mazutis, Ignas Masilionis, Ojasvi Chaudhary 2020. Frozen tissue dissociation for single-nucleus RNA-Seq. protocols.io https:// | 2021-04-15 09:14:56 | |||||
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7: User-friendly protocol: Retina Tissue Sections RNA FISH Resource Report Resource Website |
Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin | Human Cell Atlas Method Development Community | Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA; These authors contributed equally, Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA; These authors contributed equally, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA; Present address: Department of Genome Sciences, University of Washington, Seattle, WA, USA; These authors contributed equally; Correspondence: [email protected] (P.Y.), [email protected] (C.L.C.), [email protected] (B. J. B.), Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA; These authors contributed equally, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA, Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA; Correspondence: [email protected] (P.Y.), [email protected] (C.L.C.), [email protected] (B. J. B.), Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; Department of Systems Biology, Harvard Medical School, Boston, MA, USA; Correspondence: [email protected] (P.Y.), [email protected] (C.L.C.), [email protected] (B. J. B.) | http://saber.fish/ | 1 | 2020 | Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin 2020. 7: User-friendly protocol: Retina Tissue Sections RNA FISH. protocols.io https:// | 2021-04-15 09:14:57 | ||||
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Nuclei isolation from human intestinal biopsic tissue for single-cell genomic applications Resource Report Resource Website |
Ran Zhou, Oni Basu | DOI:10.17504/protocols.io.bma2k2ge | Human Cell Atlas Method Development Community, Helmsley project_Basu lab | This protocol provides an efficient method to isolate nuclei from human intestinal biopsy samples for single cell applications (RNA-seq or ATAC-seq). | University of Chicago, University of Chicago | 2 | 2020 | Ran Zhou, Oni Basu 2020. Nuclei isolation from human intestinal biopsic tissue for single-cell genomic applications. protocols.io https://dx.doi.org/10.17504/protocols.io.bma2k2ge | 2021-04-15 09:15:00 | |||
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Human adult generic tissue dissociation *in development* Resource Report Resource Website |
Anna Wilbrey-Clark, Adam Hunter | DOI:10.17504/protocols.io.39ygr7w | Human Cell Atlas Method Development Community | The aim with this protocol is to pull together what we have learnt from dissociating other tissues, to generate a 'generic' protocol that can be used on multiple tissues for single cell RNA sequencing. To date (June 2019) we have mainly used this on human adult lung and gut and it is definitely still in development! We are also testing replacing liberase TL with liberase DH. The trypsin step may or may not be necessary depending on tissue, but can liberate additional cells compared to the liberase alone. | Sanger Institute, Cambridge, HCA, Sanger Institute, Cambridge, CGaP | 1 | 2019 | Anna Wilbrey-Clark, Adam Hunter 2019. Human adult generic tissue dissociation *in development*. protocols.io https://dx.doi.org/10.17504/protocols.io.39ygr7w | 2021-04-15 09:14:59 |
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