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Authors: Brent A. Biddy, Wenjun Kong, Kenji Kamimoto, Chuner Guo, Sarah Waye, Tao Sun, Samantha Morris
Summary: Single-cell technologies are offering unprecedented insight into complex biology, revealing the behavior of rare cell populations that are typically masked in bulk population analyses. One current limitation of single-cell approaches is that lineage relationships are lost as a result of cell processing, restricting interpretations of the data collected. Elegant computational approaches have been developed in an effort to infer these missing observations, but it remains a challenge to reconstruct true reprogramming trajectories using these tools. Although sophisticated lineage tracing solutions to connect cell history with fate are emerging, these protocols are either not compatible with high-throughput scRNA-seq, or require genome editing strategies that are not readily deployed in some systems. Here, our protocol describes a single-cell resolution clonal tracking approach, ‘CellTagging’, based on combinatorial cell indexing, permitting the parallel capture of lineage information and cell identity. CellTagging integrates with high-throughput single-cell RNA-sequencing, where iterative rounds of cell labeling enable the construction of multi-level lineage trees. This straightforward lentiviral-labeling approach can be applied to an array of cell biological applications to simultaneously profile lineage and identity, at single-cell resolution.

Proper citation: Brent A. Biddy, Wenjun Kong, Kenji Kamimoto, Chuner Guo, Sarah Waye, Tao Sun, Samantha Morris 2019. Single-cell mapping of lineage and identity via CellTagging. protocols.io https://dx.doi.org/10.17504/protocols.io.xu6fnze Copy   

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Authors: Kenneth Schackart, Kattika Kaarj
Group: 481b Laboratory
Summary: Introductory protocol for cell nucelus staining using and imaging. This protocol uses Invitrogen™ NucBlue™ LiveReady Probes™.

Proper citation: Kenneth Schackart, Kattika Kaarj 2019. Mammalian Cell Nucleus Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.xswfnfe Copy   

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Authors: Na Wang, Chuan Wang, Xi'an Fu, Gongqi Yu, Zhenhua Yue, Tingting Liu

Proper citation: Na Wang, Chuan Wang, Xi'an Fu, Gongqi Yu, Zhenhua Yue, Tingting Liu 2018. Genotype with Quant Studio™ 12K Flex protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.pvbdn2n Copy   

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Authors: Elizabeth Fozo

Proper citation: Elizabeth Fozo 2020. 1. Sample_prep_WB. protocols.io https:// Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Q5U Hot Start High-Fidelity DNA Polymerase is a modified version of Q5®High-Fidelity DNA Polymerase, a novel thermostable DNA polymerase that possesses 3′ to 5′ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases.

Proper citation: New England Biolabs 2020. PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515): Amplification of bisulfite-converted, deaminated, or damaged DNA (Including FFPE DNA). protocols.io https://dx.doi.org/10.17504/protocols.io.7sdhna6 Copy   

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Authors: Jessica Sacher

Proper citation: Jessica Sacher 2017. Natural Transformation of Campylobacter jejuni. protocols.io https://dx.doi.org/10.17504/protocols.io.magc2bw Copy   

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Authors: Catherine C. Bannon, Douglas A. Campbell
Summary: We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX.

Proper citation: Catherine C. Bannon, Douglas A. Campbell 2017. Phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.jqzcmx6 Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: Published work using this protocol:- A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions. Mabou Tagne A, Marino F, Legnaro M, Luini A, Pacchetti B, Cosentino M.Int J Mol Sci. 2019 Apr 13;20(8):1833. doi: 10.3390/ijms20081833

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 06 - Culture of Human PMN - TNF-α production. protocols.io https://dx.doi.org/10.17504/protocols.io.bkrakv2e Copy   

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Authors: Latoya Allen, Marissa Ciesla, Yasin Seven, Elisa Gonzalez-Rothi, Gordon Pool
Group: SPARC
Summary: This protocol describes the procedure for retrogradely labeling bilateral phrenic motor neuron pools in the cervical spinal cord using intrapleural injections of cholera toxin B fragment

Proper citation: Latoya Allen, Marissa Ciesla, Yasin Seven, Elisa Gonzalez-Rothi, Gordon Pool 2019. SPARC Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons Using Intrapleural Injections of Cholera Toxin B Fragment. protocols.io https://dx.doi.org/10.17504/protocols.io.2kpgcvn Copy   

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Authors: Anna Behle
Group: Axmann Lab, CyanoWorld, iGEM Duesseldorf 2018
Summary: Linear assembly of PCR fragments.Can be used to quickly and efficiently fuse promoters, terminators, fusion proteins etc. without time-consuming sub-cloning steps.

Proper citation: Anna Behle 2019. Overlap extension PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.psndnde Copy   

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Authors: Bei Wang, Wen-Hsin Sandy Lee, Helen Huang, Patricia Ng, Eve Ngoh, Chia Yin Lee, Hwee Ching Tan, Rabiatul Adawiyah, Mun Kuen Soh, Frannie Teo, Yvonne Yeap, Yuanyu Hu, Cheng-I Wang
Group: Coronavirus Method Development Community

Proper citation: Bei Wang, Wen-Hsin Sandy Lee, Helen Huang, Patricia Ng, Eve Ngoh, Chia Yin Lee, Hwee Ching Tan, Rabiatul Adawiyah, Mun Kuen Soh, Frannie Teo, Yvonne Yeap, Yuanyu Hu, Cheng-I Wang 2020. IgG expression and purification. protocols.io https://dx.doi.org/10.17504/protocols.io.bitvken6 Copy   

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Authors: Kristin Evans
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryA high-fat diet of varying composition and percent fat is administered to induce obesity in mice. High-fat diet induced obesity is causally associated with insulin resistance and type 2 diabetes. Diets will be provided in consultation with the investigator needs. This service applies to specialized feeding for vendor supplied animals being supplied out of the UC Davis vendor approved barrier facility.

Proper citation: Kristin Evans 2019. UC Davis - High fat diet feeding. protocols.io https://dx.doi.org/10.17504/protocols.io.xfmfjk6 Copy   

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Authors: Phuong Thao Bui, Quentin Nunes, Yong Li, David G.Fernig
Summary: Interactions between heparan sulfate proteoglycans and the partner proteins regulate many aspects of cell functions, however, their binding properties are still equivocal. Developed eight years ago was a method for selective labelling of lysine residues in the heparin binding sites of Fibroblast Growth Factors (FGFs). The labelling of arginine residues is far more challenging, due to the multiple reaction products between a dicarbonyl and a guanidino group.

Proper citation: Phuong Thao Bui, Quentin Nunes, Yong Li, David G.Fernig 2019. Selective protection and labelling of arginine/lysine side chains in HBSs of proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.wq6fdze Copy   

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Authors: Gurdon Institute mediak
Summary: Make LB agar medium.

Proper citation: Gurdon Institute mediak 2019. Make LB agar medium. protocols.io https://dx.doi.org/10.17504/protocols.io.55zg876 Copy   

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Authors: Dania Villarnovo

Proper citation: Dania Villarnovo 2018. Phalloidin Immunofluorescence Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.rd8d29w Copy   

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Authors: Ariel Cerda, Catalina Ibarra-Henriquez, Valentina Sebastian, Grace Armijo, Liliana Lamig, Carolina Miranda, Marcela Lagos, Sandra Solari, Ana María Guzmán, Teresa Quiroga, Susan Hitschfeld, Eleodoro Riveras, Marcela Ferres, Rodrigo A. Gutiérrez, Patricia García, Aniela Wozniak
Group: Coronavirus Method Development Community, Reclone.org (The Reagent Collaboration Network)

Proper citation: Ariel Cerda, Catalina Ibarra-Henriquez, Valentina Sebastian, Grace Armijo, Liliana Lamig, Carolina Miranda, Marcela Lagos, Sandra Solari, Ana María Guzmán, Teresa Quiroga, Susan Hitschfeld, Eleodoro Riveras, Marcela Ferres, Rodrigo A. Gutiérrez, Patricia García, Aniela Wozniak 2020. Validation of Selected RNA Extraction Method. protocols.io https:// Copy   

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Authors: Collin Closek, Anni Djurhuus, Katie Pitz, Ryan Kelly, Reiko Michisaki, Kristine Walz, Hilary Starks, Francisco Chavez, Alexandria Boehm, Mya Breitbart
Group: MBON eDNA
Summary: This protocol is aimed at amplifying the cytochrome c oxidase subunit I (COI) mitochondrial gene in eukaryotes. The primers (forward: mlCOIintF, reverse: HCO2198) utilized in this protocol are based on the primers utilized in Leray et al. 2013 (forward) and Folmer et al. 1994 (reverse).Amplicons generated using this protocol can then be sequenced using the Illumina platform.Primers used:Fluidigm CS1+mlCOIinfFFluidigm CS2+HCO2198Secondary COI PCR primersPE1-BC-CS1PE2-BC-CS2

Proper citation: Collin Closek, Anni Djurhuus, Katie Pitz, Ryan Kelly, Reiko Michisaki, Kristine Walz, Hilary Starks, Francisco Chavez, Alexandria Boehm, Mya Breitbart 2018. Environmental DNA (eDNA) COI metabarcoding Illumina MiSeq NGS PCR Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mwnc7de Copy   

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Authors: New England Biolabs, Menna Teffera
Summary: This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext®Ultra II End Repair/dA-Tailing Module (NEB #E7546), for Illumina®-compatible library construction.The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) or the NEBNext Ultra II FS DNA Module (NEB #E7810).

Proper citation: New England Biolabs, Menna Teffera 2019. NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546). protocols.io https://dx.doi.org/10.17504/protocols.io.4nqgvdw Copy   

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Authors: Sarah Hessen-Schmidt

Proper citation: Sarah Hessen-Schmidt 2016. PCR HSP60 - 96 well plate. protocols.io https://dx.doi.org/10.17504/protocols.io.fbebije Copy   

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Authors: Alessandra Trojani

Proper citation: Alessandra Trojani 2019. GEP analysis of BM CD34+/lin- cells of patients with CML. protocols.io https://dx.doi.org/10.17504/protocols.io.yncfvaw Copy   

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