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| Name | Authors | DOI | Group |
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Tissue lysis and digestion for MS analysis Resource Report Resource Website |
David Kotol | DOI:10.17504/protocols.io.yguftww | Human Cell Atlas Method Development Community, Human Protein Atlas | Here, a workflow for sample preparation and quantification of brain and pancreatic tissue proteins with use of heavy labelled protein standards (QPrESTs) is described. The standards are added before the digestion ensuring high reproducibility and elimination of nuances apearing during proteolytical cleavage of proteins and further steps such as solid phase extraction. QPrESTs are heavy labelled recombinant protein fragments covering regions of proteins with low homology and span over 50-150 aminoacids. They were produced as a part of Human Protein Atlas project in auxotrofic E.coli and individually purified, serving as an excelent resource for MS-based protein quantification. | Department of Protein Science, Royal Institute of Technology, Stockholm, Sweden | 1 | 2019 | David Kotol 2019. Tissue lysis and digestion for MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.yguftww | 2021-04-15 09:15:59 | |||
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Single Cell Calling Cards Library Preparation Resource Report Resource Website |
Arnav Moudgil, Robi D. Mitra | DOI:10.17504/protocols.io.xwifpce | Human Cell Atlas Method Development Community, Transposon Calling Cards | This protocol describes how to create calling card libraries from single cell RNA. We assume you have successfully transformed cells with piggyBac self-reporting transposons and either undirected piggyBac transposase or your favorite transcription factor (YFTF) fused to piggyBac. We also assume you have optimized the dissociation protocol for your specific cells or tissues and can generate single cell suspensions. | Washington University, Saint Louis, Washington University, Saint Louis | https://doi.org/10.1101/538553 | 1 | 2019 | Arnav Moudgil, Robi D. Mitra 2019. Single Cell Calling Cards Library Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.xwifpce | 2021-04-15 09:16:02 | ||
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4: User-friendly protocol: Cost-efficient Primer Exchange Reaction (PER) concatemerization (SABER-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. 4: User-friendly protocol: Cost-efficient Primer Exchange Reaction (PER) concatemerization (SABER-FISH). protocols.io https:// | 2021-04-15 09:16:08 | ||||
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Adult Mouse Spleen Dissociation (On ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.q5cdy2w | Human Cell Atlas Method Development Community | Protocol used to dissociate adult (8-10 wk) mouse spleen into single cells. Attained >95% viability, a variety of cell sizes, and ~10 million cells from 12 mg tissue. | CCHMC | 2 | 2018 | Andrew Potter 2018. Adult Mouse Spleen Dissociation (On ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q5cdy2w | 2021-04-15 09:16:05 | |||
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Frozen Tissue Nuclei Extraction (v2) Resource Report Resource Website |
Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko | DOI:10.17504/protocols.io.7xchpiw | Human Cell Atlas Method Development Community | Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). This protocol is based strongly on a similar extraction protocol from the McCarroll lab. | Broad Institute, Broad Institute, Broad Institute, Broad Institute, Broad Institute, Broad Institute | 1 | 2019 | Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko 2019. Frozen Tissue Nuclei Extraction (v2). protocols.io https://dx.doi.org/10.17504/protocols.io.7xchpiw | 2021-04-15 09:16:07 | |||
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COVID Airway Processing for scRNAseq Resource Report Resource Website |
Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber | DOI:10.17504/protocols.io.bjj8kkrw | Human Cell Atlas Method Development Community, Coronavirus Method Development Community | This protocol describes the isolation of lymphocytes and pan-mononuclear cells from airway samples. | Columbia University, Columbia University, Columbia University, Columbia University | 1 | 2020 | Peter Szabo, Steven Wells, Peter A. Sims, Donna Farber 2020. COVID Airway Processing for scRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.bjj8kkrw | 2021-04-15 09:16:06 | |||
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Adult mouse skin dissociation protocol (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.tupenvn | Human Cell Atlas Method Development Community | This protocol was developed to dissociate adult (8-10 wk) mouse skin "on ice". It utilizes two layers of digestion with a Bacillus licheniformis protease cocktail, combined with mechanical disruption from a dounce homogenizer. The cell yield is 3000 cells/mg. | CCHMC | 1 | 2018 | Andrew Potter 2018. Adult mouse skin dissociation protocol (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.tupenvn | 2021-04-15 09:16:12 | |||
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Intestinal Organoid Dissociation and Nuclei Isolation for Single Cell ATAC-Seq Resource Report Resource Website |
Heather Eckart, Ran Zhou, Nadia Khan | DOI:10.17504/protocols.io.bmdbk22n | Human Cell Atlas Method Development Community, Helmsley project_Basu lab | This protocol provides a procedure for human intestinal organoid dissociation into a single cell suspension and nuclei isolation prior to Single Cell ATAC-Sequencing. | University of Chicago, University of Chicago, University of Chicago | 1 | 2020 | Heather Eckart, Ran Zhou, Nadia Khan 2020. Intestinal Organoid Dissociation and Nuclei Isolation for Single Cell ATAC-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bmdbk22n | 2021-04-15 09:16:15 | |||
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Adult mouse ear dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.ucwesxe | Human Cell Atlas Method Development Community | This protocol is used to dissociate adult (8-10 wk) mouse ear "on ice" (in order to reduce gene expression artifacts). The cell yield is 9000 cells/mg (213000 cells total from 23 mg tissue), with 98% viable. The protocol involves a 2-layered dissociation, incubating on ice with bacillus licheniformis enzyme. The dissociation is augmented by the use of a dounce homogenizer to help break up the tissue. | CCHMC | 1 | 2018 | Andrew Potter 2018. Adult mouse ear dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.ucwesxe | 2021-04-15 09:16:22 | |||
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CGAP MACS Live Dead Separation Resource Report Resource Website |
Adam Hunter | DOI:10.17504/protocols.io.qz5dx86 | Human Cell Atlas Method Development Community | CGAP | 1 | 2018 | Adam Hunter 2018. CGAP MACS Live Dead Separation. protocols.io https://dx.doi.org/10.17504/protocols.io.qz5dx86 | 2021-04-15 09:16:30 | ||||
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CGAP Human Lung Dissociation - Tissue Stability Study Resource Report Resource Website |
Anna Wilbrey-Clark, Adam Hunter | DOI:10.17504/protocols.io.34kgquw | Human Cell Atlas Method Development Community | Sanger Institute, Cambridge, HCA, CGAP | 1 | 2019 | Anna Wilbrey-Clark, Adam Hunter 2019. CGAP Human Lung Dissociation - Tissue Stability Study. protocols.io https://dx.doi.org/10.17504/protocols.io.34kgquw | 2021-04-15 09:16:17 | ||||
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sci-RNA-seq3 Resource Report Resource Website |
Junyue Cao, Jay Shendure | DOI:10.17504/protocols.io.9yih7ue | Human Cell Atlas Method Development Community | Single-cell combinatorial indexing (‘sci-’) is a methodological framework that employs split-pool barcoding to uniquely label the nucleic acid contents of large numbers of single cells or nuclei. Although the throughput of ‘sci-’ methods increases exponentially with the number of rounds of indexing, this potential has yet to be fully realized because of other factors such as the rate of cell loss and the limited reaction efficiency of some steps. To address this, we developed and extensively optimized 3-level sci-RNA-seq (sci-RNA-seq3), resulting in a workflow that can profile millions of cells per experiment. As previously multiple samples (e.g. replicates, timepoints, etc.) can be barcoded during the first round of indexing and concurrently processed. | University of Washington | https://www.nature.com/articles/s41586-019-0969-x#citeas | 1 | 2020 | Junyue Cao, Jay Shendure 2020. sci-RNA-seq3. protocols.io https://dx.doi.org/10.17504/protocols.io.9yih7ue | 2021-04-15 09:16:31 | ||
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RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.mepc3dn | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves.smFISH is a powerful technique because it allows the quantitative measurement of the exact copy number and spatial distribution of individual mRNAs within single cells in intact tissues. MERFISH extends these powerful abilities to the transcriptome scale by massively multiplexing smFISH measurements by encoding individual RNA species with error-robust barcodes and reading out the barcodes via a series of smFISH measurements. We anticipate this method to be relatively easily adopted by other laboratories and the techniques described in this collection should facilitate these efforts as will the supporting protocols and code that can be found at: http://zhuang.harvard.edu/merfish/. | 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 Multiplexed Error Robust Fluorescence in situ Hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.mepc3dn | 2021-04-15 09:16:35 | ||
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Scanorama Resource Report Resource Website |
Brian Hie, Bryan Bryson, Bonnie Berger | DOI:10.17504/protocols.io.9gch3sw | Human Cell Atlas Method Development Community | Scanorama enables efficient integration of heterogeneous scRNA-seq data sets. | Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology | http://scanorama.csail.mit.edu | 1 | 2019 | Brian Hie, Bryan Bryson, Bonnie Berger 2019. Scanorama. protocols.io https://dx.doi.org/10.17504/protocols.io.9gch3sw | 2021-04-15 09:16:21 | ||
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invertedClampFISH ligation Resource Report Resource Website |
Benjamin Emert | DOI:10.17504/protocols.io.qnkdvcw | Human Cell Atlas Method Development Community, RajLab | Protocol for making invertedClampFISH probes. | University of Pennsylvania | 1 | 2018 | Benjamin Emert 2018. invertedClampFISH ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.qnkdvcw | 2021-04-15 09:16:25 | |||
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Adult mouse kidney dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.rnmd5c6 | Human Cell Atlas Method Development Community | Protocol for adult (8-10 week) mouse kidney dissociation performed on ice to reduce artifact gene expression. The first layer, consisting of collagenase digestion, breaks down the tissue and releases some cells and glomeruli and tubules. The second layer consists of bacillus licheniformis digestion for 15 min. augmented with a thermomixer at 1400 RPM and passaging with a 27 gauge needle. The second layer is meant to thoroughly break up remaining tubules and glomeruli, releasing cells such as podocytes. The final yield is 250K cells from 18 mg tissue with 98% viability, approximately 14,000 cells/mg tissue. Approximately 1% of released cells are podocytes (visualized using kidneys from MAFB-GFP+ mice using a hemocytometer). | Cincinnati Children's Hospital Medical Center | 6 | 2018 | Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.rnmd5c6 | 2021-04-15 09:16:46 | |||
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Massively parallel long-read sequencing of single cell RNA isoforms Resource Report Resource Website |
Jafar S Jabbari, Luyi Tian | DOI:10.17504/protocols.io.8d9hs96 | Human Cell Atlas Method Development Community | This protocol describes the subsampling of 10x Chromium generated single cell GEMs after reverse transcription for cDNA amplification. The protocol enables transcriptome sequencing of full-length cDNA from a flexible number of single cells captured on Chromium using long-read sequencers such as Nanopore and PacBio, in addition to preparing short-read Illumina libraries. The protocol is compatible with 10x Single Cell 3' and 5' Gene Expression kits including feature barcoding and also other 10x compatible multiomics methodologies such as CITE-seq. | Peter Doherty Institute for Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia | 1 | 2019 | Jafar S Jabbari, Luyi Tian 2019. Massively parallel long-read sequencing of single cell RNA isoforms. protocols.io https://dx.doi.org/10.17504/protocols.io.8d9hs96 | 2021-04-15 09:16:26 | |||
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Embedding and freezing fresh human tissue in OCT using isopentane Resource Report Resource Website |
Kenny Roberts, Liz Tuck | DOI:10.17504/protocols.io.95mh846 | Human Cell Atlas Method Development Community | For many cutting-edge spatial transcriptomic analysis methods, it is essential to prepare and store tissue in a manner that preserves RNA integrity, tissue morphology, and spatial orientation.This protocol describes the process of freezing fresh or fixed and cryoprotected tissues in optimal cutting temperature compound (OCT) using an isopentane-dry ice slurry. Rapid controlled freezing preserves RNA and mimises ice damage, while embedding in OCT prepares tissues for future cryosectioning. | Wellcome Sanger Institute, Wellcome Sanger Institute | 3 | 2019 | Kenny Roberts, Liz Tuck 2019. Embedding and freezing fresh human tissue in OCT using isopentane. protocols.io https://dx.doi.org/10.17504/protocols.io.95mh846 | 2021-04-15 09:16:55 | |||
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Single Cell Dissociation of Fresh Lung Tissue Resource Report Resource Website |
Lance Peter, Mei-I Chung, Nicholas E. Banovich | DOI:10.17504/protocols.io.7xkhpkw | Human Cell Atlas Method Development Community | Translational Genomics Research Institute, Phoenix, AZ, Translational Genomics Research Institute, Phoenix, AZ, Translational Genomics Research Institute, Phoenix, AZ | https://www.biorxiv.org/content/10.1101/753806v1 | 1 | 2019 | Lance Peter, Mei-I Chung, Nicholas E. Banovich 2019. Single Cell Dissociation of Fresh Lung Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.7xkhpkw | 2021-04-15 09:16:55 | |||
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Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications Resource Report Resource Website |
Ran Zhou | DOI:10.17504/protocols.io.bde3i3gn | Human Cell Atlas Method Development Community, Helmsley project_Basu lab | The protocol is adapted from Fujii's and Smillies's reports for single cell transcriptome analysis from human intestines. It provides details on acquirement of single cell suspension from epithelium and lamina propria. This methods is modified to generate appropriate meterials from patient's intestinal biopsies for sinlge-cell transcriptome and genomic applications. | University of Chicago | 1 | 2020 | Ran Zhou 2020. Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications. protocols.io https://dx.doi.org/10.17504/protocols.io.bde3i3gn | 2021-04-15 09:16:28 |
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