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Isolation of nuclei from frozen tissue for ATAC-seq and other epigenomic assays Resource Report Resource Website |
Ryan Corces, William J. Greenleaf, Howard Y. Chang | DOI:10.17504/protocols.io.6t8herw | Human Cell Atlas Method Development Community | This protocol enables the isolation of nuclei from frozen tissues. These nuclei are suitable for use in ATAC-seq, single-cell ATAC-seq, ChIP-seq, HiC/3C, and many other assays. | Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, Satpathy AT, Rubin AJ, Montine KS, Wu B, Kathiria A, Cho SW, Mumbach MR, Carter AC, Kasowski M, Orloff LA, Risca VI, Kundaje A, Khavari PA, Montine TJ, Greenleaf WJ†, Chang HY†. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. 2017. Nature Methods. (PMID: 28846090). | Gladstone Institutes, Stanford University, Stanford University | https://www.ncbi.nlm.nih.gov/pubmed/28846090 | 1 | 2019 | Ryan Corces, William J. Greenleaf, Howard Y. Chang 2019. Isolation of nuclei from frozen tissue for ATAC-seq and other epigenomic assays. protocols.io https://dx.doi.org/10.17504/protocols.io.6t8herw | 2021-04-15 09:15:29 | |
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Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension Resource Report Resource Website |
Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes | DOI:10.17504/protocols.io.bjhkkj4w | Human Cell Atlas Method Development Community, Coronavirus Method Development Community | A protocol for recovering viable single cell suspensions from cryopreserved human nasopharyngeal swabs for downstream applications, such as single-cell RNA-seq. The illustrated schematic below details the process. | Boston Children's Hospital, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, University of Mississippi Medical Center, Boston Children's Hospital, Massachusetts Institute of Technology, Boston Children's Hospital | 1 | 2020 | Ying Tang, Carly G.K. Ziegler, Vincent N. Miao, Andrew W. Navia, Joshua D. Bromley, Kenneth J. Wilson, Yilianys Pride, Mohammad Hasan, Taylor Christian, Hannah Laird, Anna Owings, Meredith Sloan, Haley B. Williams, Tanya O. Robinson, George E. Abraham III, Michal Senitko, Sarah C. Glover, Bruce Horwitz, Alex K. Shalek, Jose Ordovas-Montanes 2020. Human Nasopharyngeal Swab Processing for Viable Single-Cell Suspension. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhkkj4w | 2021-04-15 09:15:29 | |||
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Healthy Control Recruitment and Enrollment Resource Report Resource Website |
Alyssa Ward, Gracie Gordon, Jimmie Ye | DOI:10.17504/protocols.io.bbkvikw6 | Human Cell Atlas Method Development Community | The Immune Cell Census aims to collect expression data from 1000 blood samples collected from healthy individuals. This protocol outlines our inclusion/exclusion criteria and enrollment process. | UCSF, UCSF, UCSF | 4 | 2020 | Alyssa Ward, Gracie Gordon, Jimmie Ye 2020. Healthy Control Recruitment and Enrollment. protocols.io https://dx.doi.org/10.17504/protocols.io.bbkvikw6 | 2021-04-15 09:15:01 | |||
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Copy of Cell preparation for scRNA-Seq from diluted bodily fluids Resource Report Resource Website |
Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire | Human Cell Atlas Method Development Community, NCIHTAN, My protocols | Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA, Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA, Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA | https://doi.org/10.1126/science.aaz2193 | 1 | 2020 | Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire 2020. Copy of Cell preparation for scRNA-Seq from diluted bodily fluids. protocols.io https:// | 2021-04-15 09:15:08 | ||||
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Single-molecule mRNA detection and counting in mammalian tissue Resource Report Resource Website |
Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker, Zi Peng Fan, Xuebing Wu, Alexander van Oudenaarden | DOI:10.17504/protocols.io.mc7c2zn | Human Cell Atlas Method Development Community | We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3–7 d, depending on the type of analysis. | Massachusetts Institute of Technology, Hubrecht Institute—KNAW, Massachusetts Institute of Technology, Weizmann Institute of Science, Massachusetts Institute of Technology, Hubrecht Institute—KNAW, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology | http://www.nature.com/doifinder/10.1038/nprot.2013.109 | 1 | 2018 | Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker, Zi Peng Fan, Xuebing Wu, Alexander van Oudenaarden 2018. Single-molecule mRNA detection and counting in mammalian tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.mc7c2zn | 2021-04-15 09:15:06 | ||
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Whole Blood Processing (SepMate) Resource Report Resource Website |
Yang Sun, David Lee, Jimmie Ye | DOI:10.17504/protocols.io.ba87ihzn | Human Cell Atlas Method Development Community | Purpose: To purify PBMCs from 20 or 50mL whole blood (per donor). SepMate tubes will be used with Lymphoprep to ease processing time and effort. | UCSF, UCSF, UCSF | 1 | 2020 | Yang Sun, David Lee, Jimmie Ye 2020. Whole Blood Processing (SepMate). protocols.io https://dx.doi.org/10.17504/protocols.io.ba87ihzn | 2021-04-15 09:15:33 | |||
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ExFISH - Tissue Slice Resource Report Resource Website |
Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden | DOI:10.17504/protocols.io.n6adhae | Human Cell Atlas Method Development Community | The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine. | Massachusetts Institute of Technology, Massachusetts Institute of Technology, University of Pennsylvania, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, University of Pennsylvania, Massachusetts Institute of Technology | http://expansionmicroscopy.org/ExFISHV1.0.docx | 1 | 2018 | Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden 2018. ExFISH - Tissue Slice. protocols.io https://dx.doi.org/10.17504/protocols.io.n6adhae | 2021-04-15 09:15:15 | ||
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ExFISH - Cultured Cells Resource Report Resource Website |
Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden | DOI:10.17504/protocols.io.n32dgqe | Human Cell Atlas Method Development Community | The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine. | Massachusetts Institute of Technology, Massachusetts Institute of Technology, University of Pennsylvania, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, University of Pennsylvania, Massachusetts Institute of Technology | http://expansionmicroscopy.org/ExFISHV1.0.docx | 1 | 2018 | Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj, Edward S Boyden 2018. ExFISH - Cultured Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n32dgqe | 2021-04-15 09:15:43 | ||
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Smart-seq2 single-cell RNA-Seq modified method Resource Report Resource Website |
John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev | DOI:10.17504/protocols.io.pbgdijw | Human Cell Atlas Method Development Community | For the past several decades, due to technical limitations, the field of transcriptomics has focused on population‐level measurements that can mask significant differences between individual cells. With the advent of single‐cell RNA‐Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome‐wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high‐throughput, single‐cell RNA‐Seq libraries. Complementary DNA (cDNA) is made from full‐length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second “template‐switch” primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing. Curr. Protoc. Mol. Biol. 107:4.22.1‐4.22.17. © 2014 by John Wiley & Sons, Inc.In single-cell RNA-Seq, small amounts of sample loss during a number of steps can lead to significant decreases in transcript detection sensitivity. A decrease in assay sensitivity results in data that is only accurate and reproducible for highly expressed genes, limiting the scope and confidence of gene expression analyses. Further complications in assay sensitivity arise from an uneven distribution of sequencing reads along a transcript; usually, in SMARTer, there is a bias towards more reads at the 3′ end of the transcript. Even coverage along a transcript improves the accuracy of analytical tools used to quantify gene expression and transcript isoform abundance. A method published by Picelli et al (Nature Methods, 2013) modified the traditional SMARTer protocol to address this by improving transcript detection, coverage, accuracy, yield, and cost. Following the same strategy as SMARTer library construction, Smart-seq2 uses several alternative reagents to generate whole-transcriptome full-length cDNA libraries. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; } Avoiding small-volume, bead-based SPRI cleanups of each sample is an effective way of reducing loss and increasing assay sensitivity. Lysing single cells in a guanidine thiocyanate buffer necessitates SPRI cleanup due to the protein denaturing effects of the compound, which will affect downstream reactions, like reverse transcription. Multiple alternative lysis buffers exist that address this. The Ambion Single Cell Lysis buffer (Life technologies, #4458235), often used for single-cell RT-PCR, only requires the addition of a stop solution to inactivate its lytic activity before subsequent reactions. A hypotonic lysis buffer with small amounts of RNase-inhibitor and surfactant, as described in Smart-seq2, is the preferred buffer due to the lack of a need for a post-lysis cleanup or the addition of a stop solution prior to reverse transcription. However, the optimal lysis strategy will depend on the experimental system being analyzed. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; } Smart-seq2 takes additional steps to minimize sample loss during library construction. The reverse transcription is improved by the addition of betaine and additional magnesium chloride to the reaction mix and by the use of a template-switch oligonucleotide with one locked nucleic acid (LNA) riboguanosine base. These improvements assist in the hybridization between the template-switch oligonucleotide and the cDNA product, thereby increasing the probability of successfully introducing a second PCR adapter onto the cDNA product (see Figure 1). A second key improvement was made in the preamplification PCR step, which can be heavily biased against either long transcripts or those containing regions with high G/C content. Picelli et al found that the preamplification PCR is improved by using the KAPA HiFi HotStart ReadyMix, which dramatically improved coverage and sensitivity, particularly for GC-rich transcripts. .justify:after { content: ""; display:inline-block; width: 100%; } | 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/ | 1 | 2018 | John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev 2018. Smart-seq2 single-cell RNA-Seq modified method. protocols.io https://dx.doi.org/10.17504/protocols.io.pbgdijw | 2021-04-15 09:15:41 | ||
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Single-Cell Dissociation of Human Trabecular Meshwork Resource Report Resource Website |
Alexi Mcadams, Tavé van Zyl | DOI:10.17504/protocols.io.bfdyji7w | Human Cell Atlas Method Development Community | Fresh trabecular meshwork tissue is dissected from post-mortem human eyes and dissociated into a single cell suspension. | Department of Ophthalmology, Harvard Medical School; Glaucoma Service, Mass. Eye and Ear, Boston, MA , Department of Ophthalmology, Harvard Medical School; Glaucoma Service, Mass. Eye and Ear, Boston, MA | 1 | 2020 | Alexi Mcadams, Tavé van Zyl 2020. Single-Cell Dissociation of Human Trabecular Meshwork . protocols.io https://dx.doi.org/10.17504/protocols.io.bfdyji7w | 2021-04-15 09:15:46 | |||
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703.3 URMC HTC_Cryopreservation_of_Isolated_Cells_061220 Resource Report Resource Website |
Gloria Pryhuber, Ravi Misra, Heidie Huyck, Gautam Bandyopadhyay | DOI:10.17504/protocols.io.biz6kf9e | Human Cell Atlas Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab | Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)Standard Operating Procedures 703.2_HTC102314rev061019Cryopreservation of Isolated CellsPurpose and Scope of the Procedure or Laboratory Assay | University of Rochester Medical Center, University of Rochester Medical Center, University of Rochester Medical Center, University of Rochester Medical Center | 1 | 2020 | Gloria Pryhuber, Ravi Misra, Heidie Huyck, Gautam Bandyopadhyay 2020. 703.3 URMC HTC_Cryopreservation_of_Isolated_Cells_061220. protocols.io https://dx.doi.org/10.17504/protocols.io.biz6kf9e | 2021-04-15 09:15:20 | |||
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Nuclei isolation from human kidney for single-nucleus RNA-seq Resource Report Resource Website |
Ben Humphreys, Yuhei Kirita | DOI:10.17504/protocols.io.nahdab6 | Human Cell Atlas Method Development Community | This protocol is based upon Habib, N et al., Nature Methods 14: 955-958, 2017 and Basu et al., Protocol Exchange 2017: DroNc-seq step-by-step with adaptions for adult human kidney including: Tissue mincing, homogenization strokes, addition of protease inhibitor and RNasin and adjustments to strainer size and sequence. | Washington University in St. Louis, The Humphreys Lab | 1 | 2018 | Ben Humphreys, Yuhei Kirita 2018. Nuclei isolation from human kidney for single-nucleus RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.nahdab6 | 2021-04-15 09:15:46 | |||
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Protocol for the isolation of non-parenchymal liver cells from human liver biopsies (1-2cm3) Resource Report Resource Website |
Charlotte Scott, Martin Guilliams | DOI:10.17504/protocols.io.viwe4fe | Human Cell Atlas Method Development Community | Optimised protocol for the isolation of non-parenchymal cells from human liver biopsies. Used on 1-2cm3 biopsies. | VIB-UGent Center for Inflammation Research, Ghent, Belgium, VIB-UGent Center for Inflammation Research, Ghent, Belgium | 1 | 2018 | Charlotte Scott, Martin Guilliams 2018. Protocol for the isolation of non-parenchymal liver cells from human liver biopsies (1-2cm3). protocols.io https://dx.doi.org/10.17504/protocols.io.viwe4fe | 2021-04-15 09:15:20 | |||
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Adult mouse lung cell dissociation (on ice) Resource Report Resource Website |
Andrew Potter | DOI:10.17504/protocols.io.q6bdzan | 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 | 1 | 2018 | Andrew Potter 2018. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q6bdzan | 2021-04-15 09:15:47 | |||
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Isolation of single nuclei from solid tissues Resource Report Resource Website |
Blue Lake, Kun Zhang | DOI:10.17504/protocols.io.ufketkw | Human Cell Atlas Method Development Community, KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can circumvent the non-uniform or incomplete dissociation of solid tissues into single cells. The isolation of nuclei can also circumvent RNA degradation or any introduction of technical artefacts (such as stress responses) that could be triggered during whole cell dissociation methods. Data generated from single-nucleus genomic assays permits discovery of molecular cell types that can be used to define the overall cellular makeup of a tissue or organ, and ultimately will inform upon adult human tissue atlases. | University of California, San Diego, University of California, San Diego | http://genome-tech.ucsd.edu/ZhangLab/ | 1 | 2019 | Blue Lake, Kun Zhang 2019. Isolation of single nuclei from solid tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.ufketkw | 2021-04-15 09:15:20 | ||
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Cell Hashing Resource Report Resource Website |
Marlon Stoeckius, Peter Smibert | DOI:10.17504/protocols.io.nfzdbp6 | Human Cell Atlas Method Development Community | This protocol is for performing Cell Hashing only. Sample multiplexing and super-loading on single cell RNA-sequencing platforms.Cell Hashing uses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct samples, which can be subsequently pooled in one scRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples. | New York Genome Center Technology Innovation Lab, New York Genome Center Technology Innovation Lab | https://cite-seq.com/cell-hashing/ | 1 | 2018 | Marlon Stoeckius, Peter Smibert 2018. Cell Hashing. protocols.io https://dx.doi.org/10.17504/protocols.io.nfzdbp6 | 2021-04-15 09:15:25 | ||
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Cell dissociation from airway biopsies with cold-active protease for single-cell RNA-seq Resource Report Resource Website |
Laure-Emmanuelle Zaragosi, Pascal Barbry | DOI:10.17504/protocols.io.x3efqje | Human Cell Atlas Method Development Community | This protocol provides details on the cell dissociation that should be performed to obtain single-cell suspensions from airway biopsies.Biopsies may come from tracheal, bronchial or nasal epithelium.Cell dissociation is performed at 4°C to avoid gene expression alterations and maximize viability. The typical cell number recovery is 40 000 cells for one biopsy.Cell suspensions are suitable for single-cell RNA-sequencing protocols. | Université Côte d'Azur, CNRS, IPMC, 06560 Valbonne, France, Université Côte d'Azur, CNRS, IPMC, 06560 Valbonne, France | 1 | 2019 | Laure-Emmanuelle Zaragosi, Pascal Barbry 2019. Cell dissociation from airway biopsies with cold-active protease for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.x3efqje | 2021-04-15 09:15:49 | |||
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3: 30mer branch melting temperatures (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. 3: 30mer branch melting temperatures (SABER-FISH). protocols.io https:// | 2021-04-15 09:15:49 | ||||
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sci-ATAC-seq3 Resource Report Resource Website |
Silvia Domcke, Andrew J. Hill, Riza M. Daza, Cole Trapnell, Darren A. Cusanovich, Jay Shendure | DOI:10.17504/protocols.io.be8mjhu6 | Human Cell Atlas Method Development Community | We developed an improved assay for single cell profiling of chromatin accessibility that both uses three levels of combinatorial indexing and, in contrast with previous iterations of sci-ATAC-seq and related methods, does not rely on molecularly barcoded Tn5 complexes (sci-ATAC-seq3). Rather, the first two rounds of indexing are achieved by ligation to either end of the conventional, uniformly loaded Tn5 transposase complex (standard Nextera™), while the final round of indexing remains through PCR. Relative to two-level sci-ATAC-seq but similar to sci-RNA-seq3, sci-ATAC-seq3 reduces the per-cell cost of library preparation as well as the rate of collisions, opening the door to experiments on the scale of 10^6 cells. This protocol no longer requires cell sorting, and we also optimized ligase and polymerase choice, kinase concentration, and oligo designs and concentrations, to maximize the number of fragments recovered from each cell. Of note, while maintaining an enrichment in accessible regions, we made the explicit choice to maximize complexity at the expense of specificity for accessible sites. In particular, we found that the fixation conditions could be tuned to adjust the sensitivity (i.e. complexity) vs. specificity (i.e. enrichment in accessible sites) of the assay. | Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA, Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA, Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA, Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA, USA, Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA; Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA; Asthma and Airway Disease Research Center, University of Arizona, Tucson, AZ, USA, Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA; Brotman Baty Institute for Precision Medicine, Seattle, WA, USA; Allen Discovery Center for Cell Lineage Tracing, Seattle, WA, USA; Howard Hughes Medical Institute, Seattle, WA, USA | 1 | 2020 | Silvia Domcke, Andrew J. Hill, Riza M. Daza, Cole Trapnell, Darren A. Cusanovich, Jay Shendure 2020. sci-ATAC-seq3. protocols.io https://dx.doi.org/10.17504/protocols.io.be8mjhu6 | 2021-04-15 09:15:27 | |||
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CGAP Dead cell removal EasySep kit with The Big Easy Magnet Resource Report Resource Website |
Adam Hunter | DOI:10.17504/protocols.io.qz6dx9e | Human Cell Atlas Method Development Community | CGAP | 1 | 2018 | Adam Hunter 2018. CGAP Dead cell removal EasySep kit with The Big Easy Magnet. protocols.io https://dx.doi.org/10.17504/protocols.io.qz6dx9e | 2021-04-15 09:15:50 |
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