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Authors: Ryan Corces, William J. Greenleaf, Howard Y. Chang
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: 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
Group: Human Cell Atlas Method Development Community, Coronavirus Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Alyssa Ward, Gracie Gordon, Jimmie Ye
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: Alyssa Ward, Gracie Gordon, Jimmie Ye 2020. Healthy Control Recruitment and Enrollment. protocols.io https://dx.doi.org/10.17504/protocols.io.bbkvikw6 Copy   

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Authors: Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire
Group: Human Cell Atlas Method Development Community, NCIHTAN, My protocols

Proper citation: Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire 2020. Copy of Cell preparation for scRNA-Seq from diluted bodily fluids. protocols.io https:// Copy   

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Authors: Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker, Zi Peng Fan, Xuebing Wu, Alexander van Oudenaarden
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Yang Sun, David Lee, Jimmie Ye
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: Yang Sun, David Lee, Jimmie Ye 2020. Whole Blood Processing (SepMate). protocols.io https://dx.doi.org/10.17504/protocols.io.ba87ihzn Copy   

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

Proper citation: 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 Copy   

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

Proper citation: 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 Copy   

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Authors: John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev
Group: Human Cell Atlas Method Development Community
Summary: 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%; }

Proper citation: 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 Copy   

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Authors: Alexi Mcadams, Tavé van Zyl
Group: Human Cell Atlas Method Development Community
Summary: Fresh trabecular meshwork tissue is dissected from post-mortem human eyes and dissociated into a single cell suspension.

Proper citation: Alexi Mcadams, Tavé van Zyl 2020. Single-Cell Dissociation of Human Trabecular Meshwork . protocols.io https://dx.doi.org/10.17504/protocols.io.bfdyji7w Copy   

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Authors: Gloria Pryhuber, Ravi Misra, Heidie Huyck, Gautam Bandyopadhyay
Group: Human Cell Atlas Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: 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

Proper citation: 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 Copy   

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Authors: Ben Humphreys, Yuhei Kirita
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Charlotte Scott, Martin Guilliams
Group: Human Cell Atlas Method Development Community
Summary: Optimised protocol for the isolation of non-parenchymal cells from human liver biopsies. Used on 1-2cm3 biopsies.

Proper citation: 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 Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability. 

Proper citation: Andrew Potter 2018. Adult mouse lung cell dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q6bdzan Copy   

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Authors: Blue Lake, Kun Zhang
Group: Human Cell Atlas Method Development Community, KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: 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.

Proper citation: Blue Lake, Kun Zhang 2019. Isolation of single nuclei from solid tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.ufketkw Copy   

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Authors: Marlon Stoeckius, Peter Smibert
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: Marlon Stoeckius, Peter Smibert 2018. Cell Hashing. protocols.io https://dx.doi.org/10.17504/protocols.io.nfzdbp6 Copy   

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Authors: Laure-Emmanuelle Zaragosi, Pascal Barbry
Group: Human Cell Atlas Method Development Community
Summary: This protocol provides details on the cell dissociation that should be performed to obtain single-cell suspensions from 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.

Proper citation: 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 Copy   

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Authors: 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
Group: Human Cell Atlas Method Development Community

Proper citation: 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:// Copy   

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Authors: Silvia Domcke, Andrew J. Hill, Riza M. Daza, Cole Trapnell, Darren A. Cusanovich, Jay Shendure
Group: Human Cell Atlas Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Adam Hunter
Group: Human Cell Atlas Method Development Community

Proper citation: 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 Copy   

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