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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.q7cdziw | 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 | 5 | 2018 | Andrew Potter 2018. Adult human small intestine cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q7cdziw | 2021-04-15 09:15:31 | |||
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RNA Imaging with MERFISH - Probe Construction Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.meqc3dw | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | Staining hundreds to thousands of RNA species each of which requires tens of unique encoding probes requires a very large number of unique oligonucleotide sequences. The sheer number of unique sequences makes traditional solid phase oligonucleotide synthesis prohibitively expensive in most cases. For example, assuming a modest cost of $0.10/base, an encoding probe length of ~100 nt, and the need for ~50,000 unique oligos for a single MERFISH measurement, the cost of the needed oligos would be $500,000! To circumvent this astronomical cost, we have developed a high-throughput approach to generating these probes which utilizes array-derived synthesis of complex oligonucleotide pools. These arrayderived complex oligonucleotide pools that contain ~100,000 custom designed sequences can be purchased for only a few thousand dollars. The challenge to using these pools is that each individual sequence is provided in quantities far too small to be used directly for labeling. Thus, we developed an enzymatic amplification protocol to generate the encoding probes in high quantity sufficient for RNA FISH experiments using the array-derived complex oligonucleotide pools as templates. | 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 - Probe Construction. protocols.io https://dx.doi.org/10.17504/protocols.io.meqc3dw | 2021-04-15 09:15:08 | ||
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invertedClampFISH ligation Resource Report Resource Website |
Benjamin Emert | DOI:10.17504/protocols.io.qnjdvcn | 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.qnjdvcn | 2021-04-15 09:15:34 | |||
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Human Kidney / Tumour Tissue Disaggregation for Single Cell RNA Sequencing (10x Genomics platform) Resource Report Resource Website |
Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy | DOI:10.17504/protocols.io.mjyc4pw | Human Cell Atlas Method Development Community | University of Cambridge, University of Cambridge, University of Cambridge, University of Cambridge | 1 | 2018 | Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy 2018. Human Kidney / Tumour Tissue Disaggregation for Single Cell RNA Sequencing (10x Genomics platform). protocols.io https://dx.doi.org/10.17504/protocols.io.mjyc4pw | 2021-04-15 09:15:08 | ||||
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Human Pancreas Histopathology Assessment Resource Report Resource Website |
Martha Campbell Thompson | DOI:10.17504/protocols.io.br7tm9nn | Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC | DRAFTPurpose:The purpose of this protocol is to outline proper procedures for fixed specimen processing using an automatic paraffin processor and sectioning and staining for histopathological review of normalcy. Scope:This protocol applies to human pancreas specimens that will be processed to paraffin blocks for HuBMAP Tissue Mapping Centers.Expected Outcome:Pancreas samples are processed properly and efficiently to whole slide images for histopathology review. Nuclei should stain blue. Cytoplasm of acinar cells should stain pink with blue zymogen granules. The large blood vessels should demonstrate clear differentiation of RBCs when present, surrounding collagen, and endothelial nuclei with scant cytoplasm. | University of Florida | 1 | 2021 | Martha Campbell Thompson 2021. Human Pancreas Histopathology Assessment. protocols.io https://dx.doi.org/10.17504/protocols.io.br7tm9nn | 2021-04-15 09:15:11 | |||
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Human Liver Caudate Lobe Dissociation for ScRNA-seq Resource Report Resource Website |
Sonya MacParland, Xue-Zhong Ma, Justin Manuel, Jeff Liu, Gary Bader, Ian McGilvray | DOI:10.17504/protocols.io.m9sc96e | Human Cell Atlas Method Development Community | One of the challenges to describing the human liver on a single cell level is the process of cell dissociation. Cell dissociation methods can be quite damaging to the cells and can lead to a biasing of the transcriptome. We have developed gentle surgical and perfusion techniques to isolate and transcriptionally profile viable resident cells from liver samples taken from deceased donor liver grafts prior to organ transplantation. | University of Toronto, University of Toronto, University of Toronto, University of Toronto, University of Toronto, University of Toronto | 2 | 2018 | Sonya MacParland, Xue-Zhong Ma, Justin Manuel, Jeff Liu, Gary Bader, Ian McGilvray 2018. Human Liver Caudate Lobe Dissociation for ScRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.m9sc96e | 2021-04-15 09:15:12 | |||
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CGAP Human Oesophagus Epithelium Dissociation - Tissue Stability Resource Report Resource Website |
Anna Wilbrey-Clark, Adam Hunter | DOI:10.17504/protocols.io.34fgqtn | Human Cell Atlas Method Development Community | Wellcome Trust Sanger Institute, HCA, CGAP | 1 | 2019 | Anna Wilbrey-Clark, Adam Hunter 2019. CGAP Human Oesophagus Epithelium Dissociation - Tissue Stability. protocols.io https://dx.doi.org/10.17504/protocols.io.34fgqtn | 2021-04-15 09:15:12 | ||||
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tCyCIF protcol (Leica BOND) version 0.1 Resource Report Resource Website |
Jia Ren Lin, Benjamin Izar, Shaolin Mei, Shu Wang, Peter Sorger | DOI:10.17504/protocols.io.rpxd5pn | Human Cell Atlas Method Development Community, Laboratory of Systems Pharmacology, Cyclic Immunofluorescence | The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics. | Laboratory of Systems Pharmacology, Harvard Medical School, Laboratory of Systems Pharmacology, Harvard Medical School, Laboratory of Systems Pharmacology, Harvard Medical School, Laboratory of Systems Pharmacology, Harvard Medical School, Laboratory of Systems Pharmacology, Harvard Medical School | 1 | 2018 | Jia Ren Lin, Benjamin Izar, Shaolin Mei, Shu Wang, Peter Sorger 2018. tCyCIF protcol (Leica BOND) version 0.1. protocols.io https://dx.doi.org/10.17504/protocols.io.rpxd5pn | 2021-04-15 09:15:13 | |||
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Human Kidney / Tumour Tissue Disaggregation for Fluorescence-activated cell sorting (bulk or single cell) and flow cytometeric analysis Resource Report Resource Website |
Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy | DOI:10.17504/protocols.io.ntfdejn | Human Cell Atlas Method Development Community | University of Cambridge, University of Cambridge, University of Cambridge, University of Cambridge | 1 | 2018 | Kevin Loudon, Alexandra Riding, John Ferdinand, Menna Clatworthy 2018. Human Kidney / Tumour Tissue Disaggregation for Fluorescence-activated cell sorting (bulk or single cell) and flow cytometeric analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ntfdejn | 2021-04-15 09:15:14 | ||||
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CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers Resource Report Resource Website |
Derek Janssens, Steven Henikoff | DOI:10.17504/protocols.io.zcpf2vn | Human Cell Atlas Method Development Community | We previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein. Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. In the manuscript attached to this version of the protocol, Meers et al. introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility; 2) a modified digestion protocol that prevents premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. Here we provide an updated CUT&RUN protocol that incorporates these enhancements, and provides three different options for the CUT&RUN MNase digestion reaction that are helpful to improve data quality or to increase throughput. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling. | Meers MP, Bryson TD, Henikoff JG, Henikoff S, Improved CUT&RUN chromatin profiling tools. eLife doi: 10.7554/eLife.46314 | Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington, USA 98109, Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington, USA 98109. | https://www.biorxiv.org/content/10.1101/569129v1 | 3 | 2019 | Derek Janssens, Steven Henikoff 2019. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. protocols.io https://dx.doi.org/10.17504/protocols.io.zcpf2vn | 2021-04-15 09:15:14 | |
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Nuclei prep for single cell RNA/ATAC seq from intestinal surgical samples Resource Report Resource Website |
Ran Zhou, Oni Basu | DOI:10.17504/protocols.io.bmbak2ie | Human Cell Atlas Method Development Community, Helmsley project_Basu lab | Isolating high-quality nuclei from intestinal surgical tissues is critical for single cell RNA/ATAC-seq. This protocol provides details on nuclei preparation for such application, with an overall working time of less than an hour, if FACS sorting is not incorporated. | University of Chicago, University of Chicago | 1 | 2020 | Ran Zhou, Oni Basu 2020. Nuclei prep for single cell RNA/ATAC seq from intestinal surgical samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bmbak2ie | 2021-04-15 09:15:44 | |||
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CUT&Tag@home Resource Report Resource Website |
Steven Henikoff, Derek Janssens, Hatice Kaya-Okur, Jorja Henikoff, Kami Ahmad | DOI:10.17504/protocols.io.bd26i8he | Human Cell Atlas Method Development Community | CUT&Tag@home uses a modification of Bench-top CUT&Tag which includes incubation in 0.1% SDS post-tagmentation for quantitative release of targeted fragments, followed directly by PCR with Triton-X100 to neutralize the SDS. This protocol is performed in single PCR tubes from nuclei to sequencing-ready libraries and should be suitable for high throughput. | Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center | 1 | 2020 | Steven Henikoff, Derek Janssens, Hatice Kaya-Okur, Jorja Henikoff, Kami Ahmad 2020. CUT&Tag@home. protocols.io https://dx.doi.org/10.17504/protocols.io.bd26i8he | 2021-04-15 09:15:16 | |||
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Dissociation of Nuclear Suspensions from Human Breast Tissues Resource Report Resource Website |
Emi Sei, Shanshan Bai, Nicholas Navin | DOI:10.17504/protocols.io.t3aeqie | Human Cell Atlas Method Development Community | This protocol can be used to prepare nuclear suspensions from fresh or frozen tissue for single cell DNA or RNA sequencing experiments. For RNA analysis it is recommended to use fresh tissue samples, due to RNA degradation that occurs during freeze- thaw cycles, however for DNA analysis either frozen or fresh tissue can be used. The protocol has been tested on breast tissues, and many frozen tumor tissues including breast, colon prostate and pancreatic samples. | University of Texas MD Anderson Cancer Center, University of Texas MD Anderson Cancer Center, University of Texas MD Anderson Cancer Center | 1 | 2018 | Emi Sei, Shanshan Bai, Nicholas Navin 2018. Dissociation of Nuclear Suspensions from Human Breast Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.t3aeqie | 2021-04-15 09:15:15 | |||
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Sampling of Human Islets for Quality Control Purposes Resource Report Resource Website |
James Lyon, Patrick Macdonald, Jocelyn Manning Fox | DOI:10.17504/protocols.io.sizecf6 | Human Cell Atlas Method Development Community | University of Alberta, University of Alberta, University of Alberta | 1 | 2019 | James Lyon, Patrick Macdonald, Jocelyn Manning Fox 2019. Sampling of Human Islets for Quality Control Purposes. protocols.io https://dx.doi.org/10.17504/protocols.io.sizecf6 | 2021-04-15 09:15:16 | ||||
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Muxab-seq protocol Resource Report Resource Website |
Yang Sun, George Hartoularos | DOI:10.17504/protocols.io.vdme246 | Human Cell Atlas Method Development Community | This protocol is for staining with AbSeq and multiplexing 16 different samples from distinct individuals into the same 10x run. | UCSF, UCSF | 1 | 2018 | Yang Sun, George Hartoularos 2018. Muxab-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.vdme246 | 2021-04-15 09:15:20 | |||
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SABER-FISH – Signal amplification for multiplexed fluorescence in situ hybridization assays 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. SABER-FISH – Signal amplification for multiplexed fluorescence in situ hybridization assays. protocols.io https:// | 2021-04-15 09:15:27 | ||||
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RNA Imaging with MERFISH - Data Analysis Resource Report Resource Website |
Jeffrey R. Moffitt, Xiaowei Zhuang | DOI:10.17504/protocols.io.metc3en | Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community | The basic decoding of MERFISH data—the identification of specific RNA species for each spot in the sample—consists of three basic steps. First, fluorescent spots must be identified in all images. Second, slight changes in the stage position between images of the same region from different hybridization rounds must be corrected. Finally, sets of fluorescent spots that occupy the same location in the sample must be decoded into binary barcodes, which, in turn, are decoded into the specific RNA species. After these RNAs have been decoded, there are a series of simple calculations that can often be performed on the measured barcodes to determine important parameters associated with the performance of the measurements, such as estimates of the binary errors made at each bit or the relative confidence that can be ascribed to the counts for each RNA species. In this section, we provide protocols for performing these basic computational tasks. Example software and data 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 MERFISH - Data Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.metc3en | 2021-04-15 09:15:25 | ||
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FR-Match: cell type matching for scRNAseq data Resource Report Resource Website |
Yun Renee Zhang, Brian Aevermann, Richard Scheuermann | DOI:10.17504/protocols.io.bmyfk7tn | Human Cell Atlas Method Development Community | FR-Match is a supervised cell phenotype matching strategy for cluster-to-cluster cell transcriptome integration across scRNAseq experiments. An R package and Shiny application are provided at https://github.com/JCVenterInstitute/FRmatch. | J. Craig Venter Institute, J. Craig Venter Institute, J. Craig Venter Institute | https://github.com/JCVenterInstitute/FRmatch | 1 | 2020 | Yun Renee Zhang, Brian Aevermann, Richard Scheuermann 2020. FR-Match: cell type matching for scRNAseq data. protocols.io https://dx.doi.org/10.17504/protocols.io.bmyfk7tn | 2021-04-15 09:15:25 | ||
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MINUTE ChIP Resource Report Resource Website |
Banushree Kumar, Simon Elsässer | DOI:10.17504/protocols.io.8nkhvcw | Human Cell Atlas Method Development Community | The need for quantitative ChIP-seq has been widely appreciated, and a number of quantitative methods have been proposed. Mint-ChIP, developed by the Bernstein lab provides a streamlined one-pot chromatin barcoding and a post ChIP linear amplification that requires only one adaptor per chromatin fragment. Here, we introduce unique molecule (unique molecular identifier [UMI]) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTEChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. For paired-end mapping, we modified the linear amplification strategy introduced in Mint-ChIP by priming the cDNA synthesis from a 30 adaptor (RA3) ligated onto the amplified RNA, thus maintaining the original genomic fragment length, retrieving atypical mononucleosomal fragment length for histone ChIP. We have also optimized the ligation conditions and stoichiometry of adaptors to chromatin to enable sequencing of the input. We show that MINUTE-ChIP is a sensitive method with a large linear dynamic range for accurately quantifying relative differences in genome-wide histone modification patterns across multiple pooled samples. | SciLife Laboratory, Karolinska Institute Stockholm, SciLife Laboratory, Karolinska Institute Stockholm | https://www.cell.com/cell-reports/fulltext/S2211-1247(19)31094-0#secsectitle0025 | 1 | 2019 | Banushree Kumar, Simon Elsässer 2019. MINUTE ChIP. protocols.io https://dx.doi.org/10.17504/protocols.io.8nkhvcw | 2021-04-15 09:15:31 | ||
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Nuclei isolation from snap frozen human pancreatic tissue using a citric acid buffer Resource Report Resource Website |
Luca Tosti, Christian Conrad | DOI:10.17504/protocols.io.tpxempn | Human Cell Atlas Method Development Community | A protocol to isolate nuclei from snap-frozen pancreatic tissue and protect RNA from RNAse-mediated degradation | Digital Health Center at BIH/Charité, Digital Health Center at BIH/Charité | 1 | 2020 | Luca Tosti, Christian Conrad 2020. Nuclei isolation from snap frozen human pancreatic tissue using a citric acid buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.tpxempn | 2021-04-15 09:15:34 |
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