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Authors: Michiyo Tsuru
Group: Clinical Proteomics and Gene Therapy Laboratory
Summary: A bone marrow aspirate was obtained from equine ilium, and mesenchymal stem cells (MSCs) were separated from the aspirate by density-gradient centrifugation (Ficoll-Hypaque gradient centrifugation) (GE Healthcare, Pittsburgh, PA, USA) and further cultured.

Proper citation: Michiyo Tsuru 2018. Culture of bone marrow stromal cells and mesenchymal stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.kcicsue 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: Monica Rieth
Group: Labyrieth
Summary: This method outlines a procedure for analyzing the growth curve of Baker's yeast, Saccharomyces cerevisiae, for the assessment of growth medias, the addition of metabolites to media, and the incorporation of heterologous genes to our yeast strain.

Proper citation: Monica Rieth 2020. Yeast growth profile analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.33jgqkn Copy   

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Authors: Wiebke Nahrendorf, Philip J Spence
Summary: Cells can stably (and heritably) alter their gene expression profile through epigenetic modifications. Histones package DNA into chromatin and can be post-translationally modified - most prominently by methylation and acetylation. These histone modifications alter chromatin structure and DNA accessibility. We optimised a protocol for reliable high quality chromatin immunoprecipitation followed by DNA sequencing (ChIPseq) starting with just 60,000 monocytes isolated directly from mouse tissues by flow sorting. Our protocol can easily be adapted to other mouse or human cell types to interrogate the genome-wide distribution of histone modifications or transcription factor binding sites in immune cells directly ex vivo.

Proper citation: Wiebke Nahrendorf, Philip J Spence 2020. low input ChIP-sequencing of immune cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bja3kign Copy   

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Authors: Jamila Nasim, Ashley Jones, Benjamin Schwessinger
Group: Team Schwessinger

Proper citation: Jamila Nasim, Ashley Jones, Benjamin Schwessinger 2020. Isolation of haustoria from stem rust Pgt 21-0 infected wheat seedlings. protocols.io https:// Copy   

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Authors: Ben Temperton
Group: Temperton Lab
Summary: Protocol for making 0.5 YTSS Agar Plates (1.5%). 

Proper citation: Ben Temperton 2017. 0.5 YTSS Agar Plates (1.5%). protocols.io https://dx.doi.org/10.17504/protocols.io.kj3cuqn Copy   

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Authors: Min Chen, minmei chen, yu ju

Proper citation: Min Chen, minmei chen, yu ju 2017. Viral RNA extraction and detection for enterovirus. protocols.io https://dx.doi.org/10.17504/protocols.io.jhzcj76 Copy   

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Authors: Jiri Hulcr
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how visualize internal structures, from mandibular mycangia, to beetle heads or bodies.This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Jiri Hulcr 2020. Microtome Dissection of Beetles. protocols.io https://dx.doi.org/10.17504/protocols.io.bnuqmevw Copy   

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Authors: Grace Kennedy, Husvinee Sundaramurthi, UCD Biomedical Facility Staff , Breandán Kennedy
Summary: Environmental factors and animal husbandry conditions are known to have implications in outcome of study results. To this end, environmental parameters (i.e. temperature, pH and conductivity) of our zebrafish facility are constantly monitored to ensure constant/limited variations to be introduced in the environment, and hence affecting our research studies. Our facility com. prises of a recirculating water system with filtration, sterilization and automatic adjustment of water parameters (pH, conductivity, temperature) with a capacity of over 4,000 litres of water, it houses over 20,000 adult fish in 1-, 3-, and 10-litre tanks. A separate nursery system houses up to 12,000 larvae aged from 6 to 30 days post fertilisation. .justify:after { content: ""; display:inline-block; width: 100%; } We have included information of our facility and key parameters measured for our zebrafish lines housed in University College Dublin for the year 2019. These conditions are applicable to all recent peer-reviewed articles published by our group. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Grace Kennedy, Husvinee Sundaramurthi, UCD Biomedical Facility Staff , Breandán Kennedy 2020. University College Dublin (UCD) Zebrafish Facility Environmental Parameters: 2019. protocols.io https://dx.doi.org/10.17504/protocols.io.bjqikmue Copy   

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Authors: George more

Proper citation: George more 2020. Avast antivirus. protocols.io https:// Copy   

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Authors: Laura Ruiz Remolina

Proper citation: Laura Ruiz Remolina 2017. LPS-induce acute lung injury (LPS nebulization, mice). protocols.io https://dx.doi.org/10.17504/protocols.io.j8ecrte Copy   

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Authors: Seoeun Lee, Lori Zeltser
Group: Optical Clearing of Tissue, SPARC
Summary: This protocol describes how to stain Brown Adipose Tissue using iDisco protocol and visualize the result via confocal microscope.

Proper citation: Seoeun Lee, Lori Zeltser 2019. iDisco immunolabeling in brown adipose tissue (BAT). protocols.io https://dx.doi.org/10.17504/protocols.io.wqmfdu6 Copy   

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Authors: Jeremy Worley*, Hongxu Ding*, Erin Bush, Peter Sims, and Andrea Califano
Summary: Since 2012, numerous single-cell RNA sequencing (scRNA-seq) technologies have been introduced. Most involve either significant startup cost, an expert operator, or expensive library preparation. We developed a plate-based library prep that incorporates pre-amplification pooling, unique molecular identifiers (UMIs), and mRNA enrichment using silanol coated magnetic beads. Our method, which builds off of Bush et al. (Nature Communications, 2017) and Picelli et al. (Nature Protocols, 2014), uses readily available equipment, is easy to automate, and costs a fraction of commercial kits. In addition, our method has been optimized to reduce barcode cross contamination that can occur in pooled pre-amplification reactions. This method allows for plate-based 3’ scRNA-seq with UMIs at relatively low cost, using standard lab equipment.NOTE (2018/08/29): we are currently testing optimizations described by Bagnoli et al. (Nature Communications, 2018). This protocol may be updated accordingly. 

Proper citation: Jeremy Worley*, Hongxu Ding*, Erin Bush, Peter Sims, and Andrea Califano 2018. scPLATE-seq: simple, easily automated single-cell 3’ transcriptome profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.s4hegt6 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: Chris Bellas
Summary: This protocol allows gapclosing of metagenomic islands which occur when mapping metagenomic reads to a phage reference genome. These gaps arise because multiple gene variants co-exist for specific points on the genome, these variants fluctuate in dominance over space and time. This means mapping metagenomic reads from one location onto a phage reference genome from another will often result in multiple metagenomic islands. These gaps can be closed with the following protocol, allowing the other gene variants present in a phage pan-genome to be detected. The protocol relies on detection of the same phages in similar ecosystems and relies on the phage being sequenced to sufficient depth in each sample to allow metagenomic assembly of large contigs (>10 X coverage). Prerequisites:Bowtie2Geneious software packageSPAdes assembler Blast+ tools

Proper citation: Chris Bellas 2020. Gapclosing metagenomic islands. protocols.io https://dx.doi.org/10.17504/protocols.io.bix8kfrw Copy   

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Authors: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma
Group: GigaScience Press
Summary: Amplification Free Paired End Library Construction Protocol.

Proper citation: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma 2020. Amplification Free Paired End Library Construction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bd3ti8nn Copy   

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Authors: New England BioLabs, Inc.
Group: New England Biolabs (NEB)
Summary: Cas9 nuclease may be used in vivo to create targeted genome modifications.  There are several ways in which to introduce Cas9-guide RNA complexes into cells.  Here we present a method for the transfection of Cas9 RNP’s into HEK293 FT cells using Thermo Fisher Lipofectamine® RNAiMAX.  This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate.

Proper citation: New England BioLabs, Inc. 2016. Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX. protocols.io https://dx.doi.org/10.17504/protocols.io.frjbm4n Copy   

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Authors: Deena Rennerfeldt, Pristinavae Manning, Joana S. Raminhos, Krystyn J. Van Vliet

Proper citation: Deena Rennerfeldt, Pristinavae Manning, Joana S. Raminhos, Krystyn J. Van Vliet 2019. Technological advancements in image processing and analysis for label-free investigations of bone marrow stromal cell behavior. protocols.io https://dx.doi.org/10.17504/protocols.io.w2sfgee Copy   

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Authors: David Schruth
Summary: The following protocol was developed using ethnomusicological universals rhythm, tone, interval, repetition, transposition, and syllable diversity in order to look for them in animal calls. The literal feature definitions themselves are merged from various musical dictionary sources. And the spectrographic examples are from bird calls and songs.

Proper citation: David Schruth 2020. Protocol for scoring animal calls on structural acoustic features prevalent in human music . protocols.io https://dx.doi.org/10.17504/protocols.io.bp5emq3e Copy   

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Authors: Leonardo Ribeiro, María Cristina Vidal Pessolani

Proper citation: Leonardo Ribeiro, María Cristina Vidal Pessolani 2020. Standard Operating Procedure for Real-Time PCR Reaction (qPCR). protocols.io https:// Copy   

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