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Authors: Fatma Ayhan, Genevieve Konopka
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: This protocol outlines our preparation of single-cell suspension from surgically acquired fresh human adult brain tissue. This method is adapted from Habib et al1.1 Habib, N. et al. Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat Methods 14, 955-958, doi:10.1038/nmeth.4407 (2017).
Proper citation: Fatma Ayhan, Genevieve Konopka 2018. Rapid Nuclei Isolation from Human Brain. protocols.io dx.doi.org/10.17504/protocols.io.scpeavn Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: This protocol describes the rapid and robust differentiation of cortical neurons from hiPSCs via induced expression of the neurogenin-2 (NGN2) transcription factor (Wang et al., 2017; Zhang et al., 2013). To begin, iPSCs with a stably integrated human or mouse neurogenin-2 transgene under a tetracycline-inducible promoter are exposed to doxycycline in neuronal induction medium (IM). Since iPSCs grow as colonies, they must be single-cell dissociated to a new plate before doxycycline treatment in order to provide the differentiating neurons adequate space to begin producing neuritic extensions.Once the cells have been partially differentiated on Matrigel, they are re-plated onto dishes coated with poly-L-ornithine (PLO) for neuronal maturation. After 3 days of doxycycline treatment, the cells are committed to neural differentiation, although at this time they may have only minor neuritic elongations. These neurites are generally well-preserved after dissociation and replating, but the longer neuritic projections present in cells differentiated past 3 days are often damaged during the splitting process. For this reason, differentiated neurons are optimally replated on day 3. At this time, differentiating neurons can also be cryopreserved for use at a later date, enabling curation of large, standardized stocks of partially differentiated neurons for future experiments.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. DIFFERENTIATION OF i3NEURONS (Basic Protocol 5). protocols.io dx.doi.org/10.17504/protocols.io.5h6g39e Copy
Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: 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/.
Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization. protocols.io dx.doi.org/10.17504/protocols.io.mepc3dn Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Genomic Editing: iPSC. protocols.io dx.doi.org/10.17504/protocols.io.yayfsfw Copy
Authors: Cortina Chen, Florian T Merkle
Group: Neurodegeneration Method Development Community
Summary: This protocol is about Preparing hPSC-derived neurons for single-cell RNA sequencing.
Proper citation: Cortina Chen, Florian T Merkle 2020. Preparing hPSC-derived neurons for single-cell RNA sequencing. protocols.io dx.doi.org/10.17504/protocols.io.bpcbmisn Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: PrimersCLYBL insertion of hNIL CLYBL insertion of mag-hNIL
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. REAGENTS AND SOLUTIONS (Support Protocol 7.2). protocols.io dx.doi.org/10.17504/protocols.io.5vag62e Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Staining i3Neurons for immunofluorescence (IF) studies is difficult due to the delicate and interconnected nature of the neuronal processes. These processes are easily disrupted in the extensive series of washes in an IF study, and initial dissociation of even a few processes often results in entire sheets of neurons lifting off the culture surface. The best ways to minimize these events are by reducing the number of total washes and by carrying out washes slowly on a tilted dish. Additionally, if possible, IF studies should only be done in neurons 10 days old or younger. After this point, neurons tend to be very delicate and even extremely gentle washing typically causes substantial cell washout.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. IMMUNOCYTOCHEMISTRY OF i3NEURONS (Support Protocol 2). protocols.io dx.doi.org/10.17504/protocols.io.5wtg7en Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Isolation of cells from human dura mater. Protocol includes tissue freezing, cutting the tissue with surgical tools, plating into a 6 well plate, placing a coverslip on top of the tissue, and adding cell culture media.
Proper citation: Andrea Argouarch 2020. Dural Cell Isolation . protocols.io dx.doi.org/10.17504/protocols.io.8ghhtt6 Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Thawing Rosettes and NPC. protocols.io dx.doi.org/10.17504/protocols.io.x9afr2e Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Lentiviral infection of neurons can be used for a variety of applications in microscopy and biochemistry, provided that the transgene is driven by promoters not silenced in iPSCs or neurons (e.g., CAG, PGK In EF-1α, not CMV, see Critical Parameters and Troubleshooting). The precise parameters for optimal viral production and supernatant collection/concentration will depend on the type of virus used, viral packaging mechanism, viral packaging cell line, and specific transgene introduced. Highly efficient results have been observed with lentivirus produced by Lenti-X HEK cells, with transgenes carried in the pLEX backbone.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. TRANSDUCTION OF i3NEURONS (Support Protocol 4). protocols.io dx.doi.org/10.17504/protocols.io.5wvg7e6 Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural rosette banking. protocols.io dx.doi.org/10.17504/protocols.io.x87frzn Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Neural aggregate formation. protocols.io dx.doi.org/10.17504/protocols.io.x84fryw Copy
Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: The basic imaging process for MERFISH involves several fluid handling steps, e.g. introduction of hybridization buffers and wash and imaging buffers, in conjunction with the collection of smFISH images and the photobleaching of the sample. While it would be possible to perform these fluid exchange and imaging steps manually, we strongly recommend the use of an integrated and automated fluid exchange and imaging approach. Here we provide protocols for how to construct a fluid handling system that should be compatible with a variety of microscopes. We also discuss the basic protocols for hybridizing readout probes to samples on the microscope, imaging the sample, and bleaching residual signal.
Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Imaging. protocols.io dx.doi.org/10.17504/protocols.io.mesc3ee Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Characterization of iPSC . protocols.io dx.doi.org/10.17504/protocols.io.x82frye Copy
Authors: Ruilin Tian, Jason Hong, Sydney Sattler, Martin Kampmann
Group: Neurodegeneration Method Development Community, KampmannLab
Proper citation: Ruilin Tian, Jason Hong, Sydney Sattler, Martin Kampmann 2019. Lentiviral transduction of iPSCs with sgRNAs and sgRNA libraries. protocols.io dx.doi.org/10.17504/protocols.io.8dfhs3n Copy
Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes splitting outgrowth of dural cell from a 6 well plate into one T75 flask for expansion.
Proper citation: Andrea Argouarch 2020. Splitting p0 (6wp) to p1 (T75). protocols.io dx.doi.org/10.17504/protocols.io.8gjhtun Copy
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, Neurodegeneration Method Development Community
Summary: These are Expansion FISH (ExFISH) protocols for super-resolution imaging of RNA structure and location with diffraction-limited microscopes.Please select the appropriate protocol depending on your sample (cells or tissue).
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 Protocols For Cells and Tissues. protocols.io dx.doi.org/10.17504/protocols.io.n7bdhin Copy
Authors: Marcos Otero-Garcia, Inma Cobos
Group: Neurodegeneration Method Development Community
Summary: - Protocol based on Krishnaswami et al., Nat Protoc. 2016, 3:499-524- Used routinely in our lab for the isolation of single nuclei from fresh frozen postmortem brains from subjects with neurodegenerative disorders and healthy controls for single-nucleus RNA-seq- Works also for isolation of nuclei from fresh frozen mouse brains - Used for isolation of all nuclei or antibody-enriched populations (i.e., NeuN+ nuclei)- We always perform and strongly recommend FACS if used for single-nucleus RNA-seq
Proper citation: Marcos Otero-Garcia, Inma Cobos 2019. Isolation of single nuclei from postmortem fresh frozen human brain and immunostaining for NeuN. protocols.io dx.doi.org/10.17504/protocols.io.6tuhenw Copy
Authors: Jeffrey R. Moffitt, Xiaowei Zhuang
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: 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/.
Proper citation: Jeffrey R. Moffitt, Xiaowei Zhuang 2018. RNA Imaging with MERFISH - Data Analysis. protocols.io dx.doi.org/10.17504/protocols.io.metc3en Copy
Authors: Daniel Gyllborg, Chika Yokota, Mats Nilsson
Group: Human Cell Atlas Method Development Community, Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: In situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved tissue. This protocol can be used to detect RNA molecules at the single cell level to aid in the identification of cell types according to their gene expression. The technique uses padlock probes to target desired genes of interest and rolling circle amplification to amplify signal for a high throughput methodolgy of spatial transcriptomics. With the use of barcode sequencing, identification of numerous genes is possible through multiplexing.
Proper citation: Daniel Gyllborg, Chika Yokota, Mats Nilsson 2019. In situ sequencing for RNA analysis in tissue sections. protocols.io dx.doi.org/10.17504/protocols.io.s8vehw6 Copy
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