Protocols   Select Another Resource Report Type

Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. True-Nuclear™ Transcription Factor Staining Protocol for 5 mL Tubes. protocols.io https://dx.doi.org/10.17504/protocols.io.exrbfm6 Copy   

•   Source:

Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Selection Kits Column Protocol - 5. protocols.io https://dx.doi.org/10.17504/protocols.io.7u3hnyn Copy   

•   Source:

Authors: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Jake E. Cariello, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: The protocol describes immunohistochemistry with anti-claudin-1, imaging, image segmentation, and image analysis methods to quantify human vagus nerve morphology.

Proper citation: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Jake E. Cariello, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_Grill_OT2-OD025340_HumanVagusNerve_Claudin1IHC_Morphology. protocols.io https://dx.doi.org/10.17504/protocols.io.6fzhbp6 Copy   

•   Source:

Authors: Philip J. Haynes, Kavit H. S. Main, Alice Pyne
Summary: This is part 1 of the "Atomic Force Microscopy of DNA and DNA-Protein Interactions" collection of protocols.Collection Abstract: Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and it’s interactions with proteins.

Proper citation: Philip J. Haynes, Kavit H. S. Main, Alice Pyne 2020. 1 Preparation of Mica Substrate. protocols.io https://dx.doi.org/10.17504/protocols.io.bnb2maqe Copy   

•   Source:

Authors: Norfitriah Mohamed Sohaimi, Mohd Hair Bejo, Abdul Rahman Omar, Aini Ideris, Nurulfiza Mat Isa

Proper citation: Norfitriah Mohamed Sohaimi, Mohd Hair Bejo, Abdul Rahman Omar, Aini Ideris, Nurulfiza Mat Isa 2019. Virus. protocols.io https://dx.doi.org/10.17504/protocols.io.7zjhp4n Copy   

•   Source:

Authors: Kelsey Miller
Group: BioLegend
Summary: Using UV-induced peptide exchange, MHC/peptide monomers can be generated with conditional Flex-T™ monomers that harbor peptides of interest in their binding grooves. These new MHC monomers are subsequently multimerized using streptavidin-fluorophore conjugates. The resulting Flex-T™ reagents can be used for staining antigen-specific T cells and flow cytometric analysis. In humans, the MHC molecules are called HLA (Human Leukocyte Antigen).

Proper citation: Kelsey Miller 2016. Flex-T™ Tetramer and Cell Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e7bbhin Copy   

•   Source:

Authors: Frank Aylward
Summary: Here is a general tutoral on how to begin annotating proteins with Hidden Markov Models. A small test set of HMMs is provided in the Git repo downloaded in the first step. 

Proper citation: Frank Aylward 2018. Introduction to protein annotation with Hidden Markov Models. protocols.io https://dx.doi.org/10.17504/protocols.io.pijdkcn Copy   

•   Source:

Authors: Christian Lawrence, Jason Best, Althea James, Shane Hurley, Mitchel Shia, Michelle Urh, Brady Hirshfeld, Nina Bakker, Hugo Perdomo
Summary: Environmental and housing conditions experienced by laboratory animals exert profound effects on their biology, physiology, and behavior.  These parameters are important and often overlooked sources of potential variation in experiments, and should be reported in peer-reviewed publications in order to promote scientific reproducibility.  To that end, here we provide a summary of the environmental conditions in zebrafish (Danio rerio) aquaculture facilities at Boston Children's Hospital (BCH).   We include data on the physico-chemical, health, and nutrition of zebrafish in three separate facilities at BCH: Karp, Enders SSB, and Enders Lobby. In this year's version, we also include new information on our sanitization protocol validation, and dietary inputs. We also provide some additional characterization of our sampling methods for water parameters and health in an updated legend. This information will be applicable to any study involving zebrafish conducted in one of these three facilities during the year of 2019.

Proper citation: Christian Lawrence, Jason Best, Althea James, Shane Hurley, Mitchel Shia, Michelle Urh, Brady Hirshfeld, Nina Bakker, Hugo Perdomo 2020. Zebrafish (Danio rerio) Environmental Summary, Aquatic Resources Program, Boston Children's Hospital 2019. protocols.io https://dx.doi.org/10.17504/protocols.io.bbjhikj6 Copy   

•   Source:

Authors: Roey Angel, Ana Lara-Rodriguez, Eva Petrova
Group: SoWa RI Anaerobic and Molecular Microbiology (public)

Proper citation: Roey Angel, Ana Lara-Rodriguez, Eva Petrova 2020. FAA MEDIA (FASTIDIOUS ANAEROBES AGAR). protocols.io https:// Copy   

•   Source:

Authors: Dr. Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocolModified from Pardee, A. B., F. Jacob, and J. Monod. 1959. The genetic control and cytoplasmic expression of "inducibility" in the synthesis of ß-galactosidase in E. coli. J. Mol. Biol. 1:165-178

Proper citation: Dr. Steven Wilhelm 2017. M9 Minimal Media. protocols.io https://dx.doi.org/10.17504/protocols.io.ibqcamw Copy   

•   Source:

Authors: Leticia Peraza Echeverría, Cecilia Rodríguez, Dalia Zapata
Summary: Continuous in vitro conidial production from Mycosphaerella fijiensis mycelium. A method for in vitro production of conidia is described. M fijiensis was grown on culture medium containing V8 juice. Mycelium was macerated until a fine suspension was produced; 2 mL was carefully spread on V8 sporulation medium. The Petri dishes were incubated for 6 days at 20 °C, under continuous cool-white fluorescent and black light. After the incubation period, 2.4 mL of 1% gelatin was added to each Petri dish, the conidia carefully dislodged with a camel hair brush. Conidial production was notably stimulated by brush harvesting mycelial cultures. After 6 days of culture the first harvest was made and the same batch of petri dishes were put in the same culture conditions to stimulate new conidial production. Significant cumulative conidial production was observed after the second harvest.

Proper citation: Leticia Peraza Echeverría, Cecilia Rodríguez, Dalia Zapata 2018. Conidial production from M. fijiensis mycelium. protocols.io https://dx.doi.org/10.17504/protocols.io.sgmebu6 Copy   

•   Source:

Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Poly-Lysine Coverslip Preparation This section describes the process of creating Poly-lysine-coated coverslips that are used for the tissue slices in the CODEX® experiment workflow.

Proper citation: Franchesca Farris, Marda Jorgensen 2019. Poly-Lysine Coverslip Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.8kthuwn Copy   

•   Source:

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   

•   Source:

Authors: Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a gel purification with a Pippin Prep (Sage Science). Using this protocol, we have been successfully sequencing the wheat stripe rust Puccinia striiformis and leaf rust Puccinia triticina with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 4 gigabases with an N50 of approximately 30 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants.

Proper citation: Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger 2019. High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification. protocols.io https://dx.doi.org/10.17504/protocols.io.5isg4ee Copy   

•   Source:

Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. True-Nuclear™ Transcription Factor Staining Protocol for 5 mL Tubes. protocols.io https://dx.doi.org/10.17504/protocols.io.bacsiawe Copy   

•   Source:

Authors: Ryoji Amamoto, Emanuela Zuccaro, Paola Arlotta, Constance L. Cepko
Summary: Thousands of frozen, archived tissues from postmortem human central nervous system (CNS) are currently available in brain banks. As single cell and single nucleus technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that transcriptional analysis of the human CNS requires cell type specificity. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

Proper citation: Ryoji Amamoto, Emanuela Zuccaro, Paola Arlotta, Constance L. Cepko 2019. FIN-Seq (Frozen Immunolabeled Nuclei Sequencing). protocols.io https://dx.doi.org/10.17504/protocols.io.zxbf7in Copy   

•   Source:

Authors: Xin Liu
Group: GigaScience Press, BGI, GIGA
Summary: From here, You can know detail methods of Hi-C assembly of the Betta splendens genome.

Proper citation: Xin Liu 2018. The pipeline of Hi-C assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.qradv2e Copy   

•   Source:

Authors: Ken Youens-Clark, Bonnie Hurwitz
Group: Biosys-analytics
Summary: This describes setting up ssh keys and configuration to avoid having to use your NetID+ 2-factor authentication every time you log into the HPC.  Windows users will need to install a free Unix-like environment that provides a terminal so as to avoid using an ssh client like Putty (for example: Cygwin (http://www.cygwin.com)).  Cygwin users must be sure to install the 'ssh' tools which are not installed by default.

Proper citation: Ken Youens-Clark, Bonnie Hurwitz 2019. Login to UA HPC. protocols.io https://dx.doi.org/10.17504/protocols.io.wvwfe7e Copy   

•   Source:

Authors: Marc T. J. Johnson, Eric J. Carpenter, Zhijian Tian, Richard Bruskiewich, Jason N. Burris, Charlotte T. Carrigan, Mark W. Chase, Neil D. Clarke, Sarah Covshoff, Claude W. dePamphilis, Patrick P. Edger, Falicia Goh, Sean Graham, Stephan Greiner, Julian M. Hibberd, Ingrid Jordon-Thaden, Toni M. Kutchan, James Leebens-Mack, Michael Melkonian, Nicholas Miles, Henrietta Myburg, Jordan Patterson, J. Chris Pires, Paula Ralph, Megan Rolf, Rowan F. Sage, Douglas Soltis, Pamela Soltis, Dennis Stevenson, C. Neal Stewart Jr, Barbara Surek, Christina J. M. Thomsen, Juan Carlos Villarreal, Xiaolei Wu, Yong Zhang, Michael K. Deyholos, Gane Ka-Shu Wong
Summary: This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6) and was originally published as part of Appendix S1 of "Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes" Marc T. J. Johnson et al. PLOS ONE, November 21, 2012. https://doi.org/10.1371/journal.pone.0050226

Proper citation: Marc T. J. Johnson, Eric J. Carpenter, Zhijian Tian, Richard Bruskiewich, Jason N. Burris, Charlotte T. Carrigan, Mark W. Chase, Neil D. Clarke, Sarah Covshoff, Claude W. dePamphilis, Patrick P. Edger, Falicia Goh, Sean Graham, Stephan Greiner, Julian M. Hibberd, Ingrid Jordon-Thaden, Toni M. Kutchan, James Leebens-Mack, Michael Melkonian, Nicholas Miles, Henrietta Myburg, Jordan Patterson, J. Chris Pires, Paula Ralph, Megan Rolf, Rowan F. Sage, Douglas Soltis, Pamela Soltis, Dennis Stevenson, C. Neal Stewart Jr, Barbara Surek, Christina J. M. Thomsen, Juan Carlos Villarreal, Xiaolei Wu, Yong Zhang, Michael K. Deyholos, Gane Ka-Shu Wong 2019. RNA Isolation from Plant Tissue Protocol 6: pBIOZOL and Qiagen RNeasy Plant Mini Kit Method. protocols.io https://dx.doi.org/10.17504/protocols.io.4rfgv3n Copy   

•   Source:

Authors: Magdalena Julkowska
Group: Salt Lab KAUST
Summary: The standard protocol to propagate TOP10 Competent cells for Heatshock transformation method

Proper citation: Magdalena Julkowska 2019. Home-made TOP10 competent heat-shock cells. protocols.io https://dx.doi.org/10.17504/protocols.io.pexdjfn Copy   

•   Source: