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Authors: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian
Group: Neurodegeneration Method Development Community, KampmannLab
Summary: This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).

Proper citation: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian 2019. iNeuron pre-differentiation & differentiation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8dths6n Copy   

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Authors: Daniel Gyllborg, Mats Nilsson
Group: Human Cell Atlas Method Development Community, Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: Protocol for multiplexed in situ sequencing in tissue sections as an image-based spatial transcriptomic method. This is the second iteration of In Situ Sequencing (HybISS: Hybridization based In Situ Sequencing) based on the principles published in Ke et al. Nature Methods, 2013 and more recently Qian et al. Nature Methods, 2019. Here we present a modified method in detection chemistry using sequencing by hybridization approach for combinatorial decoding. This results in a more robust method for detecting larger probe panels for a more high-throughput in situ sequencing method.

Proper citation: Daniel Gyllborg, Mats Nilsson 2020. HybISS: Hybridization-based In Situ Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.xy4fpyw Copy   

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Authors: Andrea Argouarch
Group: Neurodegeneration Method Development Community
Summary: Protocol includes splitting growing cell line from a T75 flask into two T150 flasks for expansion.

Proper citation: Andrea Argouarch 2020. Splitting p1 (1xT75) to p2 (2xT150). protocols.io https://dx.doi.org/10.17504/protocols.io.8gnhtve Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Cell culture . protocols.io https:// Copy   

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Authors: Jacob Marsh, Celeste Karch
Group: Neurodegeneration Method Development Community
Summary: This protocol is about Maintenance and Expansion of induced pluripotent stem cells.

Proper citation: Jacob Marsh, Celeste Karch 2020. iPSC Cell Culture – Maintenance and Expansion. protocols.io https://dx.doi.org/10.17504/protocols.io.9pwh5pe Copy   

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Authors: Cortina Chen, Iman Mali, Florian T Merkle
Group: Neurodegeneration Method Development Community
Summary: This protocol is about Differentiation of hPSCs to hypothalamic neurons.

Proper citation: Cortina Chen, Iman Mali, Florian T Merkle 2020. Differentiation of hPSCs to hypothalamic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bn8qmhvw Copy   

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Authors: Fabia Filipello, Jacob Marsh, Rj Martinez, Celeste Karch
Group: Neurodegeneration Method Development Community
Summary: This protocol outlines the derivation of Hematopoietic Progenitor Cells and differentiation of iMGLs using iPSC cultures. This protocol is modified the following papers.

Proper citation: Fabia Filipello, Jacob Marsh, Rj Martinez, Celeste Karch 2020. Differentiation of iPSC into Microglia-Like Cells (iMGL). protocols.io https://dx.doi.org/10.17504/protocols.io.bkrvkv66 Copy   

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Authors: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying
Group: Neurodegeneration Method Development Community

Proper citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. High-Performance Liquid Chromatography (HPLC). protocols.io https:// Copy   

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Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: While lipid-mediated transient transfection and expression of transgenes in hiPSCs is often quite high (> 50 %), even an optimal transfection with the best editing tools can result in very low genomic integration efficiency (Basic Protocol 4).In order to remove non-edited cells, positive selection markers such as genes coding for fluorescent proteins or antibiotic resistance are often included in the insert construct under constitutive promoters, and this protocol describes general methods for the former strategy. However, constant expression of a fluorophore interferes with immunocytochemistry, and antibiotic selection can incorrectly select for cells with multiple aberrant transgene insertions. To counter these shortcomings, we developed a platform for enrichment from heterogeneous cell populations through cell-surface affinity for magnetic streptavidin beads mediated by expression of a streptavidin binding peptide (SBP) fused to the truncated extracellular and transmembrane domains of low-affinity nerve growth factor receptor (LNGFR). The SBP tag is commonly used to facilitate co-immunoprecipitation with streptavidin-coated beads, and, with fusion to the LNGFR, it is efficiently localized to the extracellular surface where it can bind the beads more readily.This construct was developed based on one initially designed for purification of transgenic T cells in suspension (Matheson, Peden, & Lehner, 2014), and it is included in Addgene cat. no. 105842 for direct use with hNIL or excision and insertion into other constructs.Here, we extend the original protocol in order to optimize enrichment following safe harbor locus insertion in iPSCs, and we present evidence to support its use as either a positive or negative selection marker in a variety of transgenic applications. However, we have found that the SBP-LNGFR construct should be the only gene expressed under a dedicated, highly active promoter, as it appears to interfere with 2A-mediated ribosome skipping. For example, a different construct, with the reverse tetracycline transactivator (rtTA) linked to SBP-LNGFR by a T2A sequence, led to disrupted differentiation due to reduced rtTA activity (see Support Protocol 6).When structured correctly, however, SBP-LNGFR permits highly selective enrichment that does not require specialized equipment as in fluorescence-activated cell sorting (FACS) and does not require extensive tuning as in antibiotic selection. Notably, the efficiency of enrichment increases exponentially with a lower frequency of positive cells in the population. In addition, the protocol is easily modified to permit negative selection, e.g., for removing a floxed transgene with Cre recombinase or a gene knockout reporter. Furthermore, cells expressing SBP-LNGFR may be transiently labeled at the cell surface by incubating with any fluorophore conjugated to streptavidin. After washing to remove unbound fluorophore, these cells may be imaged, marked for clonal picking, or FACS-purified at an equivalent efficacy to cells constitutively expressing a fluorophore marker of insertion. Fluorescence then returns to undetectable levels within one passage and does not interfere with downstream immunocytochemistry applications.Precise, label-free editing of endogenous loci may be enriched by FACS sorting for transient expression of the components necessary for editing 1 day after transfection [i.e., using a combined gRNA, Cas9, and GFP expression plasmid with an integration plasmid expressing RFP outside of the insertion sequence or by using functionalized RNPs such as S1mplex with Qdots or fluorescent streptavidin (Carlson-Stevermer et al., 2017)]. This can considerably increase the prevalence of correctly edited cells, but the combined stress of transfection and sorting can decrease cell survival. To compensate, it is recommended to transfect and sort (2.4 × 106 cells) in triplicate and to plate cells at high density following the sort. In addition, extensive downstream cloning and genotyping is often required due to the propensity for non-homologous end joining leading to mutagenesis at the target locus rather than homology-directed repair-mediated integration of the desired insert.This protocol will continue with the assumption of integration of a transgene cassette into a safe-harbor locus as described in Basic Protocol 2. Label-free gene knockouts caused by NHEJ-mediated indel formation are typically prevalent (> 15 %), so frontline enrichment is optional for these applications. Following enrichment, small stocks of heterogeneous cell populations should be frozen, and the cells should be taken directly to Basic Protocol 4 for clonal isolation.

Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. SELECTION AND ENRICHMENT OF TRANSGENIC CELL POPULATIONS (Basic Protocol 3). protocols.io https://dx.doi.org/10.17504/protocols.io.5fvg3n6 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC Freezing. protocols.io https://dx.doi.org/10.17504/protocols.io.x8zfrx6 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC gDNA Extraction: For Screening Edited Clones. protocols.io https://dx.doi.org/10.17504/protocols.io.x8afrse Copy   

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Authors: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley
Group: Neurodegeneration Method Development Community
Summary: Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore have optimized the synthesis generation of α-syn fibrils to ensure reliable, robust results. These fibrils can be added to neurons in culture, differentiated iPSCs, or injected into mice or rats. The protocol includes steps for fibril synthesis as well as sonication for fibril fragmentaion which is a critical step for inducing formation of α-syn inclusions.

Proper citation: Vijay Singh, Marta Castellana-Cruz, Nunilo Cremades, Laura Volpicelli-Daley 2020. Generation and Sonication of α-synuclein Fibrils. protocols.io https://dx.doi.org/10.17504/protocols.io.bhhrj356 Copy   

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Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community

Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. iPSC PCR: For Screening Edited Clones. protocols.io https://dx.doi.org/10.17504/protocols.io.x8bfrsn Copy   

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Authors: Tomasz Krzywkowski, Mats Nilsson
Group: Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: This protocol is for in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. In the accompanying publication, we take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice co-cultured cells and apply following protocol to genotype PCDH X and Y homologs in human brain tissue sections. These are used as examples in the accompanying publication and custom padlock probes can be designed to allow for the targeting of own desired mRNA. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.This protocol is taken directly from the accompanying publication. Further details and background information can be found in the cited published article.

Proper citation: Tomasz Krzywkowski, Mats Nilsson 2019. Detection of single nucleotide polymorphisms with padlock probes. protocols.io https://dx.doi.org/10.17504/protocols.io.wjhfcj6 Copy   

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Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community
Summary: Standardized single cell seeding protocol for Blood-Brain Barrier (BBB) differentiation.

Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Single Cell Seeding of BBB Stem Cell Model. protocols.io https://dx.doi.org/10.17504/protocols.io.8j9hur6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.s8vehw6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.x87frzn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.x84fryw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.mesc3ee Copy   

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Authors: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru
Group: Neurodegeneration Method Development Community
Summary: We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

Proper citation: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru 2019. Systemic AAV vectors for widespread and targeted gene delivery in rodents. protocols.io https://dx.doi.org/10.17504/protocols.io.84ahyse Copy   

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